DNA sequences, recombinant DNA molecules and processes for producing human lipocortin-like polypeptides

ABSTRACT

DNA sequences, recombinant DNA molecules and hosts transformed with them which produce human lipocortin-like polypeptides and methods of making and using these products. The DNA sequences and recombinant DNA molecules are characterized in that they code on expression for a human lipocortin-like polypeptide. In appropriate hosts these DNA sequences permit the production of human lipocortin-like polypeptides useful as anti-inflammatory agents and methods in the treatment of arthritic, allergic, dermatologic, ophthalmic and collagen diseases as well as other disorders involving inflammatory processes.

This is a continuation-in-part of Ser. No. 690,146, filed Jan. 10, 1985,now U.S. Pat. No. 4,879,224 and Ser. No. 712,376 filed Mar. 15, 1985 nowU.S. Pat. No. 4,874,743 and Ser. No. 765,877,filed Aug. 14 1985, nowabandoned and Ser. No. 772,892, filed Sept. 5, 1985, now abandoned.

TECHNICAL FIELD OF THE INVENTION

This invention relates to DNA sequences, recombinant DNA molecules andprocesses for producing at least one human lipocortin.* Moreparticularly, the invention relates to DNA sequences and recombinant DNAmolecules that are characterized in that they code for at least onehuman lipocortin-like polypeptide. Accordingly, hosts transformed withthese sequences may be employed in the processes of this invention toproduce the human lipocortin-like polypeptides of this invention. Thesepolypeptides possess anti-inflammatory activity and are useful in thetreatment of arthritic, allergic, dermatologic, ophthalmic and collagendiseases.

BACKGROUND OF THE INVENTION

Arachidonic acid is an unsaturated fatty acid that is a precursor in thesynthesis of compounds, such as prostaglandins, hydroxy-acids andleukotrines, that are involved in inflammation reactions. It is releasedfrom membrane phospholipids by phospholipase A₂ activity. In response toanti-inflammatory agents, such as glucocorticoids, certain cells releaseproteins that have been characterized in vitro by their ability toinhibit phospholipase A₂. Accordingly, by inhibiting arachidonic acidproduction, lipocortins block the synthesis of prostaglandins and otherinflammatory substances, thereby reducing inflammation [F. Hirata etal., "A Phospholipase A₂ Inhibitory Protein In Rabbit NeutrophilsInduced By Glucocorticoids", Proc. Natl. Acad. Sci. U.S.A., 77, No. 5,pp. 2533-36 (1980)].

To date, several phospholipase A₂ inhibitory proteins have been studied.One of them--lipomodulin--has been characterized as an about 40,000molecular weight protein that is probably degraded by proteases in thecell to two smaller active species of about 30,000 and 15,000 molecularweight [F. Hirata et al., "Identification Of Several Species OfPhospholipase Inhibitory Protein(s) By Radioimmunoassay ForLipomodulin", Biochem. Biophys. Res. Commun., 109, No. 1, pp. 223-30(1982)]. Other experimental evidence suggests that two otherphospholipase A₂ inhibitors, macrocortin (about 15,000 molecular weight)and renocortin (two species with molecular weights of about 15,000 and30,000 respectively) may also be cleavage products of larger inhibitoryproteins such as lipomodulin [J. F. Cloix et al., "Characterization AndPartial Purification Of Renocortins: Two Polypeptides Formed In RenalCells Causing The Anti-Phospholipase-like Action Of Glucocorticoids",Br. J. Pharmac., 79. pp. 313-21 (1983); G. J. Blackwell et al.,"Macrocortin: A Polypeptide Causing The Anti-Phospholipase Effect OfGlucocorticoids", Nature, 287, pp. 147-49 (1980)].

Although lipomodulin has been isolated from rabbit neutrophil cells,macrocortin from rat macrophages and renocortin from rat renomedullaryinterstitial cells, the three proteins exhibit similar biologicalactivities, molecular weights and cross-reactivity with monoclonalantibodies against lipomodulin or macrocortin. Moreover, all are inducedby glucocorticoids. Thus, it has been suggested that these phospholipaseinhibitory proteins, or lipocortins, are closely related to each otherand are produced by cells as a general physiological mechanism ofsteroid action [B. Rothhut et al., "Further Characterization Of TheGlucocorticoid-Induced Antiphospholipase Protein `Renocortin`", Biochem.Biophys. Res. Commun., 117, No. 3, pp. 878-84 (1983)].

Recent data have also indicated that the 15,000 molecular weight speciesof lipomodulin is produced by lymphocytes in response to immunogens andacts as a glycosylation-inhibiting factor, inhibiting the glycosylationof IgE-binding factors and leading to the suppression of the IgEresponse [T. Uede et al., "Modulation Of The Biologic Activities OfIgE-Binding Factors: I. Identification Of Glycosylation-InhibitoryFactor As A Fragment Of Lipomodulin", J. Immunol., 130, No. 2, pp.878-84 (1983)].

As a result of their anti-inflammatory activities, lipocortins areuseful for the treatment of disorders involving inflammatory processes.Such disorders include arthritic, allergic, dermatologic, ophthalmic andcollagen diseases. Furthermore, the use of these proteins to treatinflammation might avoid the disadvantages now associated with presentanti-inflammatory compounds.

At present two classes of compounds are being used for anti-inflammatorytherapy: corticosteroids and nonsteroidal anti-inflammatory drugs.Corticosteroids are generally disfavored due to the severe side effectsthat may be associated with their use. These effects includehypertension, gastrointestinal bleeding, muscle weakness, cataracts andconvulsions. Thus, nonsteroidal anti-inflammatory compounds arepreferred. However, these non-steroids may also produce side effects,such as adverse effects on gastric and platelet physiology and on thecentral nervous system and hematopoiesis. In addition, mostnon-steroidal anti-inflammatory agents inhibit the production ofinflammatory substances via their effect on only one of the two pathwaysfor production of those substances, i.e., either the cyclooxygenasepathway or the lipoxygenase pathway.

In contrast, lipocortins inhibit the production of inflammatorysubstances via both pathways. Furthermore, because lipocortins are onlymediators of steroid action, it is unlikely that they will produce theside effects often associated with the use of corticosteroids. Andbecause these inhibitor proteins are natural mediators produced by thecell, they are unlikely to have the side effects usually associated withmany non-steroid anti-inflammatories.

In addition, lipocortin may affect inflammation through an alternatepathway by blocking the chemotactic response of leukocytes. It has beenshown that certain peptides whose sequences code for the tyrosinephosphorylation sites found within pp60 src and MT antigen, i.e., thesrc peptide and the MT peptide, inhibit chemotaxis of rabbit neutrophilsstimulated by a chemoattractant [F. Hirata et al., "Inhibition OfLeukocyte Chemotaxis By Glu-Glu-Glu-Glu-Tyr-Pro-Met-Glu AndLeu-Ile-Glu-Asp-Asn-Glu-Tyr-Thr-Ala-Arg-Gln-Gly", Biochem. Biophys. Res.Comm., 18, pp. 682-90 (1984)]. Lipocortin contains a similar sequencewithin its amino acid sequence (GluAsnGluGluGlnGluTyr, residue number15-21). Therefore, lipocortin may exert its anti-inflammatory effect viathis sequence by inhibiting or blocking the movement of neutrophils andmacrophages into inflammed tissue.

To date, however, human lipocortins have not been purified from cells.Furthermore, even if a procedure could be developed for the purificationof lipocortins, it is doubtful that sufficient quantities of them couldbe produced for their many clinical and commercial applications.Accordingly, processes enabling the production of human lipocortins inclinically useful amounts would be highly advantageous inanti-inflammatory therapy.

SUMMARY OF THE INVENTION

The present invention solves the problems referred to above by providingDNA sequences coding for at least one human lipocortin-like polypeptideand processes for producing such polypeptides in hosts transformed withthose DNA sequences.

The DNA sequences of this invention are selected from the groupconsisting of the cDNA inserts of λLC and λNLipo21-2, DNA sequenceswhich hybridize to these cDNA inserts and which code on expression for ahuman lipocortin-like polypeptide, and DNA sequences which code onexpression for a polypeptide coded for on expression by any of theforegoing DNA sequences. Recombinant DNA molecules containing these DNAsequences, hosts transformed with them and human lipocortin-likepolypeptides coded for on expression by them are also part of thisinvention.

The DNA sequences, recombinant DNA molecules, hosts and processes ofthis invention enable the production of human lipocortin-likepolypeptides for use in the treatment of arthritic, allergic,dermatologic, ophthalmic and collagen diseases, as well as otherdiseases, involving inflammation processes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the amino acid sequences of fragments obtained from acyanogen bromide digestion of rat phospholipase A₂ inhibitor protein.

FIG. 2 depicts the amino acid sequences of fragments obtained fromtryptic digestion of rat phospholipase A₂ inhibitor protein.

FIG. 3 shows the four pools of chemically synthesized oligonucleotideDNA probes used to screen for the lipocortin DNA sequences of theinvention.

FIG. 4 displays the nucleotide sequence of the cDNA insert of λLC whichcodes for human lipocortin. The figure also depicts the predicted aminoacid sequence of the lipocortin protein. The numbers indicated below theamino acid sequence (e.g., 1, 30 and 346) represent the first, thirtiethand last amino acids of the protein based upon the coding sequence ofthe gene.

FIG. 5 depicts in schematic outline the construction of plasmidpKK233.LIP.1 used to express in one embodiment the DNA sequences of theinvention.

FIG. 6 depicts in schematic outline the construction of plasmidpLiptrc155A used to express in one embodiment the DNA sequences of theinvention.

FIGS. 7-9 depict the construction of plasmid pBg120, a yeast expressionvector for production of human lipocortin according to one embodiment ofthis invention.

FIG. 10 depicts an SDS-polyacrylamide gel analysis of crude yeastlysates. Lane a contains purified human lipocortin made in E. coliaccording to this invention. Lane b contains extracts from yeast with noplasmid. Lane d contains extracts from yeast containing pBg120. Lanes cand e contain extracts from yeast containing other plasmids without DNAsequences encoding human lipocortin.

FIGS. 11-13 depict the construction of plasmid pSVL9109, a mammalianexpression vector for production of human lipocortin according to oneembodiment of this invention.

FIG. 14 depicts an SDS-polyacrylamide gel analysis of plasmin digestedhuman lipocortin-like polypeptide. Lanes a-f represent samples taken at0, 5, 10, 20, 40 and 120 minutes, respectiVely, after addition ofplasmin.

FIG. 15 depicts the phospholipase A₂ inhibitory activity of the plasminfragments of human lipocortin-like polypeptide after fractionation byBiogel P-60 column chromatography.

FIG. 16 depicts the amino acid sequences of fragments obtained fromtryptic digestion of human lipocortin-like polypeptide.

FIG. 17 depicts the amino acid sequences of fragments obtained fromtryptic digestion of Lipo-S and Lipo-L.

FIG. 18 depicts the phospholipase A₂ inhibitory activity of the elastasefragments of human lipocortin-like polypeptide after separation by SDSpolyacrylamide gel electrophoresis.

FIG. 19 depicts the amino acid sequences of fragments obtained bytryptic digestion of e-1, e-4 and e-5.

FIG. 20 depicts the mutagenic oligonucleotides utilized according to themethods of this invention for the production of biologically activelipocortin-like polypeptide fragments by recombinant DNA techniques.

FIG. 21 is a schematic line map of the lipocortin amino acid sequenceindicating by numbers the sites along the sequence where methionines arelocated. The bracketed numbers indicate sites where methionines can beintroduced to yield fragments according to this invention. Severalfragments of this invention and their molecular weights are depictedbelow the map.

FIG. 22 depicts an SDS-polyacrylamide gel analysis of untreated andcyanogen bromide-treated human lipocortin-like polypeptide extractedfrom Lipo 8, showing a 26 Kd fragment.

FIG. 23 depicts, in Panel A, the absorbance profile at 280 nm oflipocortin (peak I) and N-lipocortin (peak II) after fractionation viaMono S high resolution cation exchange chromatography. Panel B depictsthe phospholipase A₂ inhibitory activity of the eluted lipocortin andN-lipocortin of the invention in % inhibition.

FIG. 24 depicts the tryptic maps of the lipocortin and N-lipocortinproteins isolated according to the methods of this invention.

FIG. 25 depicts the tryptic map of the N-lipocortin of the invention andcorrelates the peaks of the map with the amino acid sequences of peptidefragments contained in those peaks.

FIG. 26 depicts the four pools of chemically synthesized oligonucleotideDNA probes used to screen for the N-lipocortin DNA sequences of theinvention.

FIG. 27 displays the nucleotide sequence of the cDNA insert of pNLipl.

FIG. 28 compares the nucleotide sequence of lipocortin and theN-lipocortin cDNA sequences of this invention. The "X" on the figureindicates the end of the coding sequence and the start of the 3'non-coding sequence of the genes.

DETAILED DESCRIPTION OF THE INVENTION

In order that the invention herein described may be more fullyunderstood, the following detailed description is set forth.

In the description the following terms are employed:

Nucleotide--A monomeric unit of DNA or RNA consisting of a sugar moiety(pentose), a phosphate, and a nitrogenous heterocyclic base. The base islinked to the sugar moiety via the glycosidic carbon (1' carbon of thepentose) and that combination of base and sugar is called a nucleoside.The base characterizes the nucleotide. The four DNA bases are adenine("A"), guanine ("G"), cytosine ("C"), and thymine ("T"). The four RNAbases are A, G, C, and uracil ("U").

DNA Sequence--A linear array of nucleotides connected one to the otherby phosphodiester bonds between the 3' and 5' carbons of adjacentpentoses.

Codon--A DNA sequence of three nucleotides (a triplet) which encodesthrough mRNA an amino acid, a translation start signal or a translationtermination signal. For example, the nucleotide triplets TTA, TTG, CTT,CTC, CTA and CTG encode for the amino acid leucine ("Leu"), TAG, TAA andTGA are translation stop signals and ATG is a translation start signal.

Reading Frame--The grouping of codons during the translation of mRNAinto amino acid sequences. During translation the proper reading framemust be maintained. For example, the DNA sequence GCTGGTTGTAAG may beexpressed in three reading frames or phases, each of which affords adifferent amino acid sequence:

GCT GGT TGT AAG--Ala-Gly-CysLys

G CTG GTT GTA AG--Leu-Val-Val

GC TGG TTG TAA G--Trp-Leu-(STOP)

Polypeptide--A linear array of amino acids connected one to the other bypeptide bonds berween the α-amino and carboxy groups of adjacent aminoacids.

Peptidase--An enzyme which hydrolyzes peptide bonds.

Genome--The entire DNA of a cell or a virus. It includes inter alia thestructural gene coding for the polypeptides of the substance, as well asoperator, promoter and ribosome binding and interaction sequences,including sequences such as the ShineDalgarno sequences.

Gene--A DNA sequence which encodes through its template or messenger RNA("mRNA") a sequence of amino acids characteristic of a specificpolypeptide.

Transcription--The process of producing mRNA from a gene or DNAsequence.

Translation--The process of producing a polypeptide from mRNA.

Expression--The process undergone by a gene or DNA sequence to produce apolypeptide. It is a combination of transcription and translation.

Plasmid--A nonchromosomal double-stranded DNA sequence comprising anintact "replicon" such that the plasmid is replicated in a host cell.When the plasmid is placed within a unicellular organism, thecharacteristics of that organism may be changed or transformed as aresult of the DNA of the plasmid. For example, a plasmid carrying thegene for tetracycline resistance (TET^(R)) transforms a cell previouslysensitive to tetracycline into one which is resistant to it. A celltransformed by a plasmid is called a "transformant".

Phage or Bacteriophage--Bacterial virus many of which consist of DNAsequences encapsidated in a protein envelope or coat ("capsid").

Cosmid--A plasmid containing the cohesive end ("cos") site ofbacteriophage λ. Cosmids may, because of the presence of the cos site,be packaged into λ coat protein and used to infect an appropriate host.Because of their capacity for large fragments of foreign DNA, cosmidsare useful as cloning vehicles.

Cloning Vehicle--A plasmid, phage DNA, cosmid or other DNA sequencewhich is able to replicate in a host cell, characterized by one or asmall number of endonuclease recognition sites at which such DNAsequences may be cut in a determinable fashion without attendant loss ofan essential biological function of the DNA, e.g., replication,production of coat proteins or loss of promoter or binding sites, andwhich contain a marker suitable for use in the identification oftransformed cells, e.g., tetracycline resistance or ampicillinresistance. A cloning vehicle is often called a vector.

Cloning--The process of obtaining a population of organisms or DNAsequences derived from one such organism or sequence by asexualreproduction.

Recombinant DNA Molecule or Hybrid DNA--A molecule consisting ofsegments of DNA from different genomes which have been joined end-to-endoutside of living cells and able to be maintained in living cells.

Expression Control Sequence--A sequence of nucleotides that controls andregulates expression of genes when operatively linked to those genes.They include the lac system, the β-lactamase system, the trp system, thetac and trc systems, the major operator and promoter regions of phage λ,the control region of fd coat protein, the early and late promoters ofSV40, promoters derived from polyoma virus and adenovirus,metallothionine promoters, the promoter for 3-phosphoglycerate kinase orother glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5,the promoters of the yeast α-mating factors, and other sequences knownto control the expression of genes of prokaryotic or eukaryotic cellsand their viruses or combinations thereof. For mammalian cells the genecan be linked to a eukaryotic promoter such as that for the SV40 earlyregion coupled to the gene encoding dihydrofolate reductase andselectively amplified in Chinese hamster ovary cells to produce a cellline containing many copies of actively transcribed eukaryotic genes.

Lipocortin-Like Polypeptide--A polypeptide displaying a biological orimmunological activity of a lipocortin. This polypeptide may includeamino acids in addition to those of a native lipocortin or it may notinclude all of the amino acids of native lipocortin. Finally, it mayinclude an N-terminal methionine. Lipocortin is also referred to asphospholipase inhibitor protein.

The present invention relates to DNA sequences and recombinant DNAmolecules coding for human lipocortin-like polypeptides and processesfor the production of those polypeptides.

Although a variety of selection and DNA cloning techniques mightpotentially have been employed in our isolating and cloning of a DNAsequence of this invention, in one embodiment of the invention, weadopted a selection strategy based upon rat phospholipase A₂ inhibitorprotein. Accordingly, we purified a rat phospholipase A₂ inhibitorprotein from the extracellular supernatant of rat peritoneal exudatecells and determined the amino acid sequence of various fragments ofthat protein. Based on those protein sequences, we then synthesizedseveral antisense oligonucleotide DNA probes corresponding to thoseregions of purified rat protein which had minimal nucleotide degeneracy.We then used these probes to screen a human cDNA library comprisingE.coli cells containing human macrophage cDNA sequences inserted into aphage cloning vector.

For screening, we hybridized the oligonucleotide probes to the humancDNA library utilizing a plaque hybridization screening assay and weselected clones hybridizing to a number of our probes. After isolatingand subcloning the selected human cDNA inserts into plasmids, wedetermined their nucleotide sequences and compared them to our aminoacid sequences from peptides of purified rat lipocortin. As a result ofthis comparison, we found that the nucleotide sequences of all clonesisolated coded for amino acid sequences that had a marked homology tothe amino acid sequences of our purified rat lipocortin. (Compare FIGS.1 and 2 with FIG. 4.) We confirmed that at least one of the clonesisolated contained the full length sequence encoding human lipocortin.

The cDNA sequences of this invention can be operatively linked toexpression control sequences and used in various mammalian or othereukaryotic or prokaryotic host cells to produce the human lipocortinlikepolypeptides coded for by them. For example, we have constructed highlevel expression vectors for the production of a 37 Kd human lipocortin.

In addition, the cDNA sequences of this invention are useful as probesto screen human cDNA libraries for other sequences coding forlipocortinlike polypeptides. The cDNA sequences of this invention arealso useful as probes to screen human genomic DNA libraries to selecthuman genomic DNA sequences coding for lipocortin-like polypeptides.These genomic sequences, like the above cDNA sequences of thisinvention, are then useful to produce the lipocortinlike polypeptidescoded for by them. The genomic sequences are particularly useful intransforming mammalian cells to produce human lipocortin-likepolypeptides.

According to a second embodiment of the invention, we isolated fromhuman placenta a human lipocortin-like polypeptide which showsstructural similarity, i.e., amino acid homology, to the 37 Kdrecombinant lipocortin of this invention. This protein also displaysphospholipase A₂ inhibitory activity. We have designated the protein,N-lipocortin. Lipocortin and N-lipocortin are discrete proteins thathave been defined chemically by tryptic mapping. We determined the aminoacid sequences of various fragments of N-lipocortin and based on thesesequences, we synthesized antisense oligonucleotide DNA probes. We usedthese probes to screen a human cDNA library comprising E.coli cellscontaining human placenta cDNA sequences inserted into a phage cloningvector. We isolated several clones which hybridized to the probes. Afterintroducing the cDNA insert of one of these clones into a plasmid, wedetermined the nucleotide sequence of the cDNA insert. It codes for aportion of N-lipocortin.

This cDNA sequence is useful as a probe to screen human cDNA librariesfor other sequences coding for N-lipocortin-like polypeptides. Forexample, this cDNA insert can be used to screen the human placenta cDNAlibrary of this invention for the full length sequence encodingN-lipocortin. Alternatively, the oligonucleotide probes that were usedto obtain the partial N-lipocortin cDNA sequence of this invention areuseful as probes to rescreen the placenta library for the full lengthsequence encoding N-lipocortin. In addition, the N-lipocortin cDNAsequences obtained or the probes described herein may be used to isolateother portions of the N-lipocortin gene and the full length genereconstructed by standard techniques in the art. Finally, the probes orcDNA sequences may be used as primers to synthesize full length codingsequences.

The N-lipocortin cDNA sequences of this invention are also useful asprobes to screen human genomic DNA libraries to select human genomic DNAsequences coding for N-lipocortin-like polypeptides. These genomicsequences, like the N-lipocortin cDNA sequences, are then useful toproduce N-lipocortinlike polypeptides in hosts transformed with thosesequences.

Another embodiment of the present invention relates to the production ofbiologically active human lipocortin-like polypeptide fragments whichallow better characterization of the active site of the lipocortinprotein and the design of molecules having optimal therapeutic value.Accordingly, we have generated such fragments by treatment of thelipocortin-like polypeptides of this invention with various proteases.We have also generated such biologically active fragments by recombinantDNA techniques.

The human lipocortin-like polypeptides produced by the methods of thisinvention are useful as anti-inflammatory agents and inanti-inflammatory methods and therapies. For example, such compositionsmay comprise an amount of a lipocortin-like polypeptide of thisinvention which is pharmaceutically effective to reduce inflammation anda pharmaceutically acceptable carrier. Such therapies generally comprisea method of treating patients in a pharmaceutically acceptable mannerwith those compositions.

METHODS AND MATERIALS

A wide variety of host/cloning vehicle combinations may be employed incloning or expressing the human lipocortin-like polypeptide DNAsequences prepared in accordance with this invention. For example,useful cloning or expression vehicles may consist of segments ofchromosomal, non-chromosomal and synthetic DNA sequences, such asvarious known derivatives of SV40 and known bacterial plasmids, e.g.,plasmids from E.coli including colE1, pCR1, pBR322, pMB9 and theirderivatives; wider host range plasmids, e.g., RP4, phage DNAs, e.g., thenumerous derivatives of phage λ, e.g., NM 989; and other DNA phages,e.g., M13 and filamentous single-stranded DNA phages and vectors derivedfrom combinations of plasmids and phage DNAs such as plasmids which havebeen modified to employ phage DNA or other expression control sequencesor yeast plasmids such as the 2μ plasmid or derivatives thereof.

Within each specific cloning or expression vehicle, various sites may beselected for insertion of the human lipocortin-like polypeptide DNAsequences of this invention. These sites are usually designated by therestriction endonuclease which cuts them and are well recognized bythose of skill in the art. Various methods for inserting DNA sequencesinto these sites to form recombinant DNA molecules are also well known.These include, for example, dG-dC or dA-dT tailing, direct ligation,synthetic linkers, exonuclease and polymerase-linked repair reactionsfollowed by ligation, or extension of the DNA strand with DNA polymeraseand an appropriate single-stranded template followed by ligation. It is,of course, to be understood that a cloning or expression vehicle usefulin this invention need not have a restriction endonuclease site forinsertion of the chosen DNA fragment. Instead, the vehicle could bejoined to the fragment by alternative means.

Various expression control sequences may also be chosen to effect theexpression of the DNA sequences of this invention. These expressioncontrol sequences include, for example, the lac system, the β-lactamasesystem, the trp system, the tac system, the trc system, the majoroperator and promoter regions of phage λ, the control regions of fd coatprotein, the promoter for 3-phosphoglycerate kinase or other glycolyticenzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters ofthe yeast α-mating factors, promoters for mammalian cells such as theSV40 early promoter, adenovirus late promoter and metallothioninepromoter, and other sequences known to control the expression of genesof prokaryotic or eukaryotic cells or their viruses and variouscombinations thereof. In mammalian cells, it is additionally possible toamplify the expression units by linking the gene to that fordihydrofolate reductase and applying a selection to host Chinese hamsterovary cells.

For expression of the DNA sequences of this invention, these DNAsequences are operatively-linked to one or more of the above-describedexpression control sequences in the expression vector. Such operativelinking, which may be effected before or after the chosen humanlipocortin DNA sequence is inserted into a cloning vehicle, enables theexpression control sequences to control and promote the expression ofthe DNA sequence.

The vector or expression vehicle and, in particular, the sites chosentherein for insertion of the selected DNA fragment and the expressioncontrol sequence employed in this invention, are determined by a varietyof factors, e.g., number of sites susceptible to a particularrestriction enzyme, size of the protein to be expressed, expressioncharacteristics such as the location of start and stop codons relativeto the vector sequences, and other factors recognized by those of skillin the art. The choice of a vector, expression control sequence, andinsertion site for a particular lipocortin sequence is determined by abalance of these factors, not all selections being equally effective fora given case.

It should also be understood that the DNA sequences coding for thelipocortin-like polypeptides of this invention which are inserted at theselected site of a cloning or expression vehicle may include nucleotideswhich are not part of the actual gene coding for the desired lipocortinor may include only a fragment of the entire gene for that protein. Itis only required that whatever DNA sequence is employed, a transformedhost will produce a lipocortin-like polypeptide. For example, thelipocortin-related DNA sequences of this invention may be fused in thesame reading frame in an expression vector of this invention to at leasta portion of a DNA sequence coding for at least one eukaryotic orprokaryotic carrier protein or a DNA sequence coding for at least oneeukaryotic or prokaryotic signal sequence, or combinations thereof. Suchconstructions may aid in expression of the desired lipocortin-relatedDNA sequence, improve purification or permit secretion, and preferablymaturation, of the lipocortin-like polypeptide from the host cell. Thelipocortin-related DNA sequence may alternatively include an ATG startcodon, alone or together with other codons, fused directly to thesequence encoding the first amino acid of a mature native lipocortinlikepolypeptide. Such constructions enable the production of, for example, amethionyl or other peptidyl-lipocortin-like polypeptide that is part ofthis invention. This N-terminal methionine or peptide may then becleaved intra- or extra-cellularly by a variety of known processes orthe polypeptide used together with the methionine attached to thepeptide in the anti-inflammatory compositions and methods of thisinvention.

The cloning vehicle or expression vector containing the lipocortin-likepolypeptide coding sequences of this invention is employed in accordancewith this invention to transform an appropriate host so as to permitthat host to express the lipocortin-like polypeptide for which the DNAsequence codes.

Useful cloning or expression hosts include strains of E. coli, such asE.coli W311OI^(Q), E.coli JA221, E.coli C600, E.coli ED8767, E.coli DH1,E.coli LE392, E.coli HB101, E.coli X1776, E.coli X2282, E.coli MRCI, andstrains of Pseudomonas, Bacillus, and Streptomyces, yeasts and otherfungi, animal hosts, such as CHO cells or mouse cells, other animal(including human) hosts, plant cells in culture or other hosts.

The selection of an appropriate host is also controlled by a number offactors recognized by the art. These include, for example, compatibilitywith the chosen vector, toxicity of proteins encoded by the hybridplasmid, susceptibility of the desired protein to proteolyticdegradation by host cell enzymes, contamination or binding of theprotein to be expressed by host cell proteins difficult to remove duringpurification, ease of recovery of the desired protein, expressioncharacteristics, biosafety and cost. A balance of these factors must bestruck with the understanding that not all host vector combinations maybe equally effective for either the cloning or expression of aparticular recombinant DNA molecule.

It should be understood that the human lipocortin-like polypeptides(prepared in accordance with this invention in those hosts) may includepolypeptides in the form of fused proteins (e.g., linked to aprokaryotic, eukaryotic or combination N-terminal segment to directexcretion, improve stability, improve purification or improve possiblecleavage of the N-terminal segment), in the form of a precursor oflipocortin-like polypeptides (e.g., starting with all or parts of alipocortin-like polypeptide signal sequence or other eukaryotic orprokaryotic signal sequences), in the form of a mature lipocortin-likepolypeptide, or in the form of a met-lipocortin-like polypeptide.

One particularly useful form of a polypeptide in accordance with thisinvention, or at least a precursor thereof, is a mature lipocortin-likepolypeptide with an easily cleaved amino acid or series of amino acidsattached to the amino terminus. Such construction allows synthesis ofthe protein in an appropriate host, where a start signal that may not bepresent in the mature lipocortin is needed, and then cleavage in vivo orin vitro of the extra amino acids to produce mature lipocortin-likepolypeptides. Such methods exist in the art. See, e.g., U.S. Pat. Nos.4,332,892, 4,338,397, and 4,425,437. The polypeptides may also beglycosylated, like some native lipocortins, unglycosylated, or have aglycosylation pattern different than that of native lipocortins. Suchglycosylation will result from the host cell or post-expressiontreatment chosen for the particular lipocortin.

The polypeptides of this invention also include biologically activepolypeptide fragments derived from lipocortin-like polypeptides bytreatment with proteases or by expression of a fragment of the DNAsequence which codes for a lipocortin-like polypeptide. The polypeptidesof the invention also include lipocortin-like polypeptides that arecoded for on expression by DNA sequences characterized by differentcodons for some or all of the codons of the present DNA sequences. Thesesubstituted codons may code for amino acids identical to those coded forby the codons replaced but result in higher yield of the polypeptide.Alternatively, the replacement of one or a combination of codons leadingto amino acid replacement or to a longer or shorter lipocortin-likepolypeptide may alter its properties in a useful way (e.g., increase thestability, increase the solubility or increase the therapeuticactivity).

In order that this invention may be better understood, the followingexamples are set forth. These examples are for purposes of illustrationonly and are not to be construed as limiting the scope of the inventionin any manner.

EXAMPLES A. PURIFICATION OF A RAT PHOSPHOLIPASE A₂ INHIBITOR PROTEIN

We injected male Wistar rats (200-250 kg) subcutaneously with 0.1 ml ofthe glucocorticoid, dexamethasone phosphate (1.25 mg/kg rat) in 0.9%NaCl to induce production of phospholipase A₂ inhibitor protein. We thensacrificed the rats one hour after injection by intra-cardiac injectionwith Euthasate and washed the peritoneal cavities with 10 ml ofphosphate buffered saline (50 mM KH₂ PO₄, pH 7.3, 150 mM NaCl containing2 U/ml heparin and 50 μM phenylmethylsulfonylfluoride). After we clearedthe lavages of cells and other particulate matter by centrifugation inan International centrifuge at top speed for 30 min, we assayed thecombined supernatants for lipocortin by measuring the inhibition ofrelease of labeled oleic acid from autoclaved E.coli membranes in thepresence of the supernatant and porcine pancreatic phospholipase A₂.

We performed this in vitro assay as follows: We mixed 200 μl samplesfrom the peritoneal exudate supernatant in 1.5 ml Eppendorf tubes with50 μl of 0.7M Tris-HCl (pH 8.0), 60 mM CaCl₂ buffer on ice. We thenadded 50 μl of diluted porcine pancreatic phospholipase A₂ (Catalogueno. P9139, Sigma Chemicals) and mixed and incubated the solutions on icefor 1 h. Dilutions of the phospholipase A₂ suspension into buffer (70 mMTris-HCl (pH 8.0), 6 mM CaCl₂) containing 2.5 mg/ml bovine serum albumin(BSA) were such that the final concentrations of phospholipase and BSAwere 100 ng/50 μl and 125 μg/50 μl, respectively. We then added 25 μl ofautoclaved ³ H-oleic acid-labeled E.coli as substrate and incubated themixtures at 6° C. for 8 min (both the temperature and length ofincubation must be determined for each batch of E.coli utilized).

We prepared the substrate ³ H-oleic acid-labeled E.coli as follows: Wegrew an overnight culture of E.coli in tryptone medium (1%bactotryptone, 0.5% NaCl) diluted it 1:20 with fresh broth and monitoredcell growth with a Klett meter. At a reading of 40 (i.e., when cellswere growing well), we added a 1:100 dilution of Brij 35(polyoxyethylene-23-ether, Sigma Chemicals, 10% solution in water) and a1:200 dilution of ³ H-oleic acid (9,10-³ H-[N]oleic acid, New EnglandNuclear) at 10 mCi/ml. After 5 h, when cell growth leveled off, weautoclaved the suspension for 20 min at 120° C. and stored the flaskovernight at 4° C. We then pelleted the bacteria by centrifugation for30 min at 16,000 rpm in an SS34 rotor at 4° C. and combined the loosepellets into a single tube. We washed the bacteria four times, or untilcounts in the supernatant were low, with suspension buffer (0.7MTris-HCl (pH 8.0), 10 mM CaCl₂) plus 0.1% BSA. We stored the bacteria at4° C. in suspension buffer containing 0.2% sodium azide. Typically, weprepared a 400 ml culture labeled with 20 mCi of ³ H-oleic acid. Thisyielded about 7×10⁸ cpm or about 10% of the input counts in labeledbacteria. For each point in an assay, we used 100,000 cpm, which wasadded in a volume of 25 μl. Immediately prior to use, we washed ouraliquots first in 200 mM Tris-HCl (pH 8.0), 12 mM EDTA (left on ice 30min) and then in 25 mM Tris-HCl (pH 8.0).

After the brief incubation of substrate (autoclaved labeled E.coli) withinhibitor plus phospholipase A₂, the reaction was stopped immediately byadding 100 μl of 2N HCl to each tube followed by the addition of 100 μlof 20 mg/ml delipidated BSA (99% albumin, Sigma Chemicals). Tubes werevortexed and incubated on ice for 30 min. The latter step was crucialfor extracting the lipase digestion products from the particulatemembranes.

We then pelleted the E.coli in an Eppendorf centrifuge for 5 min at10,000 g and counted 250 μl of each supernatant in 4 ml of ascintillation cocktail compatible with aqueous solutions. In this assay,we tested each sample in duplicate using an internal control in whichthe sample plus E.coli substrate was incubated both in the presence andabsence of added phospholipase. This in vitro assay demonstrated thatour peritoneal exudates contained phospholipase inhibitory activity.

To purify the lipocortin from the abovedescribed peritoneal exudatesupernatant, we first added additional protease inhibitors to thesupernatant. These typically included aprotinin (20 μg/ml), soybeantrypsin inhibitor (20 μg/ml) and EGTA (ethyleneglycol-bis-(aminoethylether) N,N'-tetraacetic acid) (0.5 mM). We incubated the exudate at 37°C. for 1 h in the presence of 0.1 U/ml calf intestinal alkalinephosphatase and concentrated it two-fold by ultrafiltration to a finalprotein concentration of 5 mg/ml using an Amicon apparatus (PM10membrane). We next dialyzed the supernatant overnight at 4° C. against40 volumes of 20 mM Tris-HCl (pH 8.1) and subjected it to DE52 ionexchange column chromatography (Whatman Ltd., column dimensions: 1 cmdia.×17 cm). Prior to use, we had equilibrated the DE52 resin with 25 mMTris-HCl (pH 8.1). We collected the flow-through fractions andconcentrated them an additional 25-fold by Amicon ultrafiltration (PM10membrane). We then subjected the concentrate to a gel filtration column(P150 resin) in 25 mM Tris-HCl (column dimensions 2.5 cm dia.×40 cm) andmonitored the column fractions for protein using absorbance at 280 nmand using the phospholipase inhibitory activity assay described above.We detected peak activity at 35-40,000 molecular weight.

We lyophilized these high activity fractions, dialyzed them against 25mM Tris-HCl (pH 6.8) containing 0.2% SDS and analyzed them using apreparative SDS-polyacrylamide gel (main gel: 15% acrylamide, 0.08%methylene bisacrylamide; stacking gel: 7.6% acrylamide, 0.21% methylenebisacrylamide). The gel analysis yielded four major protein bands.According to a modification of the Western blotting technique [H. Towbinet al., "Electrophoretic Transfer Of Proteins From Polyacrylamide GelsTo Nitrocellulose Sheets", Proc. Natl. Acad. Sci. USA, 76. pp. 4350-54(1979)] using a horse radish peroxidase antibody conjugate to visualizethe immunoreactive species, we found that only one of the four majorbands cross reacted with a neutralizing antibody which we preparedagainst a snake venom lipocortin. Accordingly, we excised this region ofthe gel, electroeluted and precipitated the contained protein from itwith 20% trichloroacetic acid and pelleted the protein by centrifugationfor 20 min at 10,000 g. After washing the pellets twice with 5 ml of-20° C. acetone, each washing being followed by a centrifugation step,we dried the pellets under vacuum.

We then digested the protein either with cyanogen bromide or withtrypsin. When utilizing cyanogen bromide digestion, we digested thepellets containing approximately 100 μg protein with 200 mg/ml ofcyanogen bromide in the dark for 16 h at 25° C. in 0.5 ml of 70% formicacid. We then diluted the reaction mixture 15-fold with water andlyophilized it. When utilizing tryptic digestion, we first resuspendedthe pellets in 0.1M NH₄ HCO₃ plus 0.1 mM CaCl₂, carboxymethylated themixture with iodoacetic acid and then incubated it with trypsin for 24 hat 37° C. During this incubation, we added trypsin three times to afinal concentration of 1.5% of total protein at time zero, 2.5% after 4h and 3.5% after 19 h.

We resolved the cleavage fragments from these digestions by highpressure liquid chromatography using a C8 column (Brownlee RP-3) for thecyanogen bromide digestion products and using a C18 column(Spectraphysics) for the tryptic digestion products, utilizing in bothcases a gradient of acetonitrile from 0-75% in 0.1% trifluoroacetic acidto elute bound fragments. We then subjected the peak fractions tosequence analysis using a gas phase sequencer (Applied Biosystems 470A).PITH-amino acids were analyzed by high pressure liquid chromatography ona 5 μm cyanocolumn (Hypersil), using a gradient of acetonitrile:methanol(4:1) from 15-55% in 0.02M sodium acetate (pH 5.7).

FIG. 1 shows the amino acid sequences of the fragments produced bycyanogen bromide digestion of our purified rat lipocortin. Of six majorpeaks, only three yielded unique sequences (CNBr 1, 2 and 3). Thesesequences are shown at the bottom of FIG. 1. Of the remaining peaks, two(CNBr 5 and 6) contained mixtures of fragments and thus could not besequenced, and peak 4 was a column artifact from which no protein wasdetected.

FIG. 2 shows the amino acid sequences of fragments from trypticdigestion. Although tryptic digestion produced over forty peaks, theamino acid sequences of only nine fractions are shown in FIG. 2. Ininstances where peaks contained more than one peptide, the appropriatefractions were subjected to a second chromatography step. T22a and T22bare sequences derived from the two components of peak 22 which wereresolved when peak 22 was rechromatographed on the same column but at aneutral pH.

B. SYNTHESIS OF OLIGONUCLEOTIDE DNA PROBES FOR LIPOCORTIN PROTEINSEQUENCES

Having determined the amino acid sequences of various regions of a ratlipocortin (see FIGS. 1 and 2), we chemically synthesized four pools ofantisense oligonucleotide DNA probes that coded for some of thoseprotein sequences (see FIG. 3). We decided to synthesize the four poolsshown in FIG. 3 because they corresponded to regions of the ratlipocortin that have minimal nucleic acid degeneracy. For each aminoacid sequence, we synthesized mixtures of probes complementary to allpossible codons. Furthermore, we synthesized the probes such that theywere complementary to the DNA sequences which code for the amino acidsequence, i.e., the probes were antisense, to enable the probes torecognize the corresponding sequences in mRNA as well as in DNA. Theamino acid sequences of the four selected regions of the rat lipocortinand all the possible nucleotide codon combinations that encode them areshown in FIG. 3. Coding degeneracies are indicated as follows: N=C, T,A, or G; R=A or G; Y=C or T; and H=A, C, or T.

Two pools of the probes, derived from sequences in the tryptic fragmentsT22a and T24 of FIG. 2, are 20-mers with 48 and 256 fold degeneracies,respectively. The other two probe pools are 17-mers with 64 and 128 folddegeneracies. To reduce further the degeneracies in the probes, we alsoprepared each pool in subpools, e.g., we prepared the 48 fold degenerate20-mer of T22a in three subpools of 16 and synthesized the other probesin four subpools. The probes in each pool were end-labeled with ³² Pusing [γ]-³² P-ATP and T4 polynucleotide kinase.

To test if our synthetic probes actually recognized human sequences, wehybridized the four subpools of T24 to GeneScreen filters containingpoly (A)⁺ mRNA from the human macrophage cell line U937, which had beeninduced with 10⁻⁷ M PMA[4β-phorbol 12βmyristate 13α-acetate] and 10⁻⁵dexamethasone, utilizing the Northern blotting technique [H. Lehrach etal., Biochemistry, 10, pp. 4743-51 (1977)]. Subpools 2 and 3 of T24hybridized to the mRNA and were detected as an 1800 base pair band uponautoradiography.

C. CONSTRUCTION AND SCREENING OF A HUMAN cDNA LIBRARY

We constructed a human cDNA library from poly (A)⁺ mRNA isolated fromhuman macrophage cell line U937. The cDNA sequences were inserted intoλgt10 and amplified in E.coli C600 hfl cells.

1. EXTRACTION OF RNA FROM HUMAN U937 CELLS

We induced human macrophage U937 cells in culture with dexamethasone(10⁻⁵ M) and phorbol ester (10⁻⁷ M) and resuspended pellets containing1.2×10⁹ cells in 48 ml lysis buffer (0.2M Tris-HCl (pH 8.0), 0.1M LiCl,25 mM EDTA, 1% SDS) plus 5 mM vanadyl complex (Bethesda Research Labs)by vortexing. We lysed the cells by addition of 24 ml phenol andvortexed for 5 min. We added 24 ml chloroform to the lysis mixture whichwas then shaken for 10 min. We separated the organic and aqueous phasesby centrifugation in a clinical centrifuge at room temperature for 10min. We reextracted the aqueous phase two times with phenol:chloroform(1:1), then two times with chloroform only. We next ethanol-precipitatedthe nucleic acids in 0.3M sodium acetate at -20° C. overnight andpelleted the nucleic acid at 14 k rpm in a Sorvall RC2B centrifuge (SS34rotor) at 4° C. for 20 min. We resuspended the pellets in 5 ml of 0.3Msodium acetate buffer, and ethanol-precipitated the nucleic acid againas described above. We resuspended the final pellet in 300 μl H₂ O andstored it at -20° C. This RNA preparation was enriched for poly(A)⁺ RNAby passage over an oligo(dT)-cellulose column (PL Biochem).

2. CONSTRUCTION OF A U937 cDNA-λgt10 LIBRARY cDNA Synthesis

We synthesized cDNA from 20 μg poly (A)⁺ mRNA isolated as describedabove. We diluted the poly (A) mRNA to 500 μg/ml in H₂ O, heated it to65° C. for 3 min, quick cooled it in a dry ice-propanol bath and thenthawed it. The RNA was then added to a reaction mixture composed of 0.1MTris-HCl (pH 8.3) at 42° C., 0.01M MgCl₂, 0.01M DTT, 1 mM dCTP, 1 mMdGTP, 1 mM dTTP, 0.5 mM dATP and 100 μCi α-³² P-ATP (3000 Ci/mmole,Amersham or New England Nuclear), 20 μg oligo (dT)₁₂₋₁₈ (PL Biochem),0.03M β-mercaptoethanol, 5 mM Vanadyl Ribonucleoside Complex (BethesdaResearch Labs), 169 U AMV Reverse Transcriptase (Seikagaku America).Final volume of the reaction mixture was 200 μl. We incubated thismixture for 2 min at room temperature and 6 h at 44° C. We terminatedthe reaction by addition of 1/10 vol 0.5M Na₂ EDTA (pH 8.0).

We adjusted the reaction mixture to 0.15M NaOH and incubated the mixtureat 37° C. for 12 h followed by neutralization with 1/10 vol 1M Tris-HCl(pH 8.0) and HCl. This was extracted with phenol:chloroform saturated TEbuffer (10 mM Tris-HCl (pH 7.0) and 1 mM Na₂ EDTA). The aqueous phasewas chromatographed through a 5 ml sterile plastic pipet containing a7×29 cm bed of Sephadex G150 in 0.01M (pH 7.4), 0.4M NaCl, 0.01M Na₂EDTA, 0.05% SDS. We pooled the front peak minus tail and precipitatedthe cDNA with 2.5 vol 95% ethanol at -20° C. This reaction yielded 1 μgof single-stranded cDNA.

Double Strand Synthesis

We resuspended the single-stranded cDNA in 200 μl (final vol) 0.1M Hepes(pH 6.9), 0.01M MgCl₂, 0.0025M DTT, 0.07M KCl, 1 mM dXTPs and 75 UKlenow fragment DNA polymerase 1 (Boehringer-Mannheim) and incubated thereaction mixture at 14° C. for 21 h. Reaction was terminated by additionof Na₂ EDTA (pH 8.0) to 0.0125M, extracted with phenol:chloroform, as inthe first cDNA step, and the aqueous phase was chromatographed on a G150column in 0.01M Tris-HCl (pH 7.4), 0.1M NaCl, 0.01M Na₂ EDTA, 0.05% SDS.We again pooled the radioactive peak minus the tail andethanol-precipitated the DNA.

We then incubated the DNA obtained with 42 U reverse transcriptase in 50μl 1 0.1M Tris-HCl (pH 8.3), 0.01M MgCl₂, 0.01M DTT, 0.1M KCl, 1 mMdXTPS, 0.03M β-mercaptoethanol for 1 h at 37° C. to completedouble-strand synthesis. The reaction was terminated and processed asdescribed above.

We cleaved the hairpin loop formed during double strand synthesis asfollows: We redissolved the pellet in 50 μl 0.03M sodium acetate (pH4.5), 0.3M NaCl, 0.003M ZnCl₂ and treated it with 100 U S₁ nuclease(Sigma) for 30 min at room temperature. Reaction was terminated byaddition of EDTA and processed as described above. The yield after S₁treatment was 900 ng dsDNA.

To assure blunt ends following S₁ nuclease digestion, we treated the DNAwith Klenow in 0.01M Tris-HCl (pH 7.4), 0.01M MgCl₂ 1 mM DTT, 0.05MNaCl, 80 μM dXTP and 12.5 U Klenow in 60 μl for 90 min at 14° C.extracted with 50:50 phenol:chloroform, and chromatographed the DNA on aG50 spin column (1 ml syringe) in 0.01M Tris-HCl (pH 7.4), 0.1M NaCl,0.01M EDTA, 0.05% SDS.

We next methylated the dsDNA by treating the DNA with EcoRI methylase in30 μl final vol 0.1M Tris-HCl (pH 8.0), 0.01M Na₂ EDTA, 24 μg BSA,0.005M DTT, 30 μM S-adenosylmethionine and 5 U EcoRI Methylase for 20min at 37° C. The reaction was heated to 70° C. for 10 min, cooled,extracted with 50:50 phenol:chloroform and chromatographed on a G50 spincolumn as described above.

We ligated the 900 ng cDNA to phosphorylated EcoRI linkers (New EnglandBiolabs) using the following conditions: 0.05M Tris-HCl (pH 7.8), 0.01MMgCl₂, 0.02M DTT, 1 mM ATP, 50 μg/ml BSA, 0.5 μg linker, 300 U T4 DNAligase in 7.5 μl final volume for 32 h at 14° C.

We adjusted the reaction to 0.1M Tris-HCl (pH 7.5), 0.05M NaCl, 5 mMMgCl₂, 100 μg/ml BSA, 125 U EcoRI (New England Biolabs), incubated themixture for 2 h at 37° C., extracted with 50:50 phenol:chloroform andchromatographed the DNA on a G50 spin column as described earlier.

We redissolved cDNA in 100 μl 0.01M Tris-HCl (pH 7.5), 0.1M NaCl, 1 mMEDTA and chromatographed it on a 1×50 cm Biogel A₅₀ (BIORAD) columnwhich had been extensively washed in the same buffer (to remove ligationinhibitors). Aliquots of fractions were run on a 1% agarose gel in TBEbuffer (0.089M Tris-HCl, 0.089M boric acid and 2.5 mM Na₂ EDTA), driedand exposed at -70° C. overnight. We pooled all fractions that werelarger than 500 base pairs and ethanol-precipitated the DNA for cloninginto an EcoRI-cut λgt10 cloning vector. The size fractionation columnyielded 126 ng of cDNA, average size approximately 1500 bp.

Library Construction

We incubated 5 μg EcoRI-cut λgt10 with 20 ng cDNA and T4 DNA ligasebuffer at 42° C. for 15 min to anneal cos sites, followed bycentrifugation for 5 sec in an Eppendorf centrifuge and addition of ATPto 1 mM and 2400 U T4 DNA ligase (New England Biolabs) in a final vol of50 μl. [See Huynh, Young and Davis, "Constructing And Screening cDNALibraries in λgt10 And λgt11", in DNA Cloning A Practical Approach (D.Glover, ed.), IRL Press (Oxford 1984).] The ligation was incubated at14° C. overnight. We packaged the λgt10 cDNA ligation mixture into phageparticles using an Amersham packaging mix [Amersham packaging protocol]and diluted with 0.5 ml SM buffer (100 mM NaCl, 10 mM MgSO₄, 50 mMTris-HCl (pH 7.5) and 0.01% gelatin).

We next infected E.coli C600 hfl cells with these phage particles toform a cDNA library of 1×107 independent recombinants [see T. Maniatis,et al., Molecular Cloning, p. 235 (Cold Spring Harbor 1982)].

For plating and amplification of the library, 1 ml of cells plus 250 μlpackaging mix was incubated at room temperature for 15 min, diluted to50 ml in LB plus MgSO₄ top agarose at 50° C. and plated on LB Mg Nuncplates. This represented a plaque density of 2×10⁵ plate. The plateswere incubated at 37° C. for approximately 8 h until plaques were nearlytouching.

We flooded the plates with 50 ml of cold SM buffer (0.01M Tris-HCl (pH7.5), 0.01M MgCl₂, 0.1 mM Na₂ EDTA) and eluted on a gyro-rotary shakerovernight at 4° C. We pooled the eluants into 250 ml bottles and spun at6 k for 10 min in a Sorvall GSA rotor. We treated the supernatants withan equal volume of cold 20% PEG 4000-2M NaCl in ice for 3 h and pelletedthe phages by centrifugation at 4 k for 30 min in an H4000 rotor in anRC-3B Sorvall centrifuge. The phage pellets were thoroughly drained,resuspended in 60 ml SM, and spun at 10,000 rpm in a SS34 rotor toremove debris. The supernatants were adjusted to 3.5M CsCl by additionof 7 g CsCl to 10 ml supernatant. We obtained phage bands bycentrifugation in a 70.1 Beckman rotor at 50,000 rpm for 18 h at 15° C.We pooled the phage bands and stored them at 4° C. for library stock.The titer obtained was 2.2×10¹³ PFU/ml.

Screening Of The Library

We screened the library with our labeled oligonucleotide probes, pools 2and 3, for lipocortin sequences using the plaque hydridization screeningtechnique of Woo [S. L. C. Woo, "A Sensitive And Rapid Method ForRecombinant Phage Screening", in Methods In Enzymology, 68, pp. 389-96(Academic Press 1979)].

An overnight culture of C600 hfl cells in L broth and 0.2% maltose waspelleted and resuspended in an equal volume of SM buffer. Wepre-adsorbed 0.9 ml of cells with 2×10⁵ phage particles at roomtemperature for 15 min. We diluted the suspension to 50 ml in LB plus 10mM MgSO₄ and 0.7% agarose at 55° C. and plated it on LB Mg Nunc plates.We screened 10 such plates. We incubated the plates at 37° C. forapproximately 8 h until plaques were nearly touching. We then chilledthe plates at 4° C. for 1 h to allow the agarose to harden. We presoakedGeneScreen Plus filters in a 1:10 dilution of the overnight E.coli C600hfl cells for 10 min at room temperature so that a lawn of E.coli cellscovered each filter. We then transferred the λ phage particles from theplaque library plates to these bacteria-coated filters as follows:

We placed the filters onto the plates containing the recombinant plaquesfor 5 min, and then lifted and incubated the filters with thephage-containing side up on LB+10 mM MgSO₄ plates at 37° C. for 5 h.

These filters were then lysed by placing them onto a pool of 0.5N NaOHfor 5 min, then neutralized on 1M Tris-HCl (pH 7.0), submerged into 1MTris-HCl (pH 7.0) and scrubbed clean of cell debris.

We prehybridized and hybridized the filters to the oligonucleotideprobes 2 and 3 in 0.2% polyvinyl-pyrrolidone (M.W. 40,000), 0.2% ficoll(M.W. 40,000), 0.2% bovine serum albumin, 0.05M Tris-HCl (pH 7.5), 1Msodium chloride, 0.1% sodium pyrophosphate, 1% SDS, 10% dextran sulfate(M.W. 500,000) and denatured salmon sperm DNA (≧100 μg/ml) according tomanufacturer's specifications (New England Nuclear) for plaque screenmembranes). We detected hybridizing λ-cDNA sequences by autoradiography.

By means of this technique, we picked 20 positive plaques and rescreenedat lower density using the same probes.

We isolated the DNA of these clones, digested with EcoRI, and hybridizedthem with the four pools of rat lipocortin probes using the Southernblot technique [E. M. Southern, "Detection Of Specific Sequences AmongDNA Fragments Separated By Gel Electrophoresis", J. Mol. Biol., 98. pp.503-18 (1975)]. Two of the clones, λ9-111 and λ4-211, contained insertedcDNA which hybridized not only to the T24 probe but to the T22a and T29probes as well.

We restricted the DNAs of these phages with EcoRI and isolated the cDNAinserts. By restricting Clone 9-111 with EcoRI we obtained a 1400 basepair fragment while restriction of Clone 4-211 gave three EcoRIfragments, 1300, 300 and 75 base pairs in length. We subcloned some ofthese fragments into plasmid pUC13 to produce recombinant plasmidspL9/20 (9-111), pL4/10 large (4-211, 1300 bp), and pL4/10 small (4-211,300 bp). We then sequenced these plasmids by the method of Maxam andGilbert [A. M. Maxam and W. Gilbert, "A New Method For Sequencing DNA",Proc. Natl. Acad. Sci. USA, 74, pp. 560-64 (1977)]. This sequencinganalysis demonstrated that the clones contained nucleotide sequenceswhich corresponded to the amino acid sequences of the purified ratlipocortin but seemed to be lacking the most 5' sequence.

A 480 base pair EcoRI-BglII fragment of pL9/20 was used as a probe torescreen the U937-λgt10 library. Seventy-two positives were isolated andpartially plaque purified by rescreening at lower density. The DNA ofeach of these positives was digested with HhaI and analyzed by theSouthern blotting technique [E. M. Southern, supra] using a 30oligonucleotide sequence (lipo 16) as a probe. Lipo 16 corresponds tothe sequence starting at base pair 81-111 of the sequence presented inFIG. 4. Fourteen of these clones showed a positive signal and werefurther analyzed by genomic sequencing [G. Church and W. Gilbert, Proc.Natl. Acad. Sci. USA, 81, p. 1991 (1984)] by digesting DNA with MspI andusing lipo 16 as probe. Seven clones, λL110, λL106, λL112, λLC, λLH, λN,and λLDD, contained an 81 base pair sequence 5' to the lipo 16 probesequence.

These clones contain cDNA sequences having an uninterrupted open readingframe that can code for 363 amino acids (see FIG. 4). We believe thatthe initiating ATG codon for lipocortin may be the ATG located atnucleotides 52-54 of FIG. 4. However, the DNA sequence of our clone,reported in FIG. 4, may be lacking one or more codons coding for aminoacids at the N-terminal end of native lipocortin. These potentialmissing codons may be isolated, if necessary, by one of skill in the artusing conventional hybridization conditions from our libraries, or otherlibraries, of genomic DNA and cDNA using as probes our clones, or morepreferably portions of the 5' terminal end of those clones. Full lengthclones may then be prepared using conventional ligation techniques andour lipocortin coding clones.

We confirmed that clone λLC of FIG. 4 contains the full length gene forlipocortin. To confirm that the ATG at nucleotides 52-54 in the λLC cDNA(FIG. 4) is the first in-frame methionine codon and thus the initiatingmethionine, we determined the 5' sequence of the lipocortin mRNA byprimer extension. A 27 oligonucleotide (lipo 18) homologous to thesequence 10 to 37 of λLC was labelled with ³² P-(γ)-ATP and hybridizedto human placental poly (A)⁺ RNA. Using this oligonucleotide as a primerand AMV reverse transcriptase, we transcribed a 60 base pair fragment ofthe most 5' end of the lipocortin mRNA. This fragment was gel purifiedand sequenced by the Maxam and Gilbert sequencing technique (supra). Theresulting sequence showed 37 base pairs homologous to sequence 1 to 37of λLC and 23 additional nucleotides that represented the 5' end of thelipocortin mRNA.

To exclude the possibility that the mRNA was in fact longer than ourprimer extension indicated, but instead had a strong stop signal forreverse transcriptase which we mistook for the 5' end, we determined theexact size of the mRNA. An oligonucleotide (lipo 17) that is homologousto sequence 94 to 128 of λLC was hybridized to placental poly (A)⁺ RNAand digested with RNase H. RNase H digests RNA only when in a hybridwith DNA and thus it introduced a defined cleavage in the mRNA at thesite where lipo 17 hybridized. This RNA was then separated on asequencing gel, blotted onto GeneScreen and probed with ³² P-labelledlipo 18. This enabled us to determine the exact size of the 5' end ofthe lipocortin mRNA, which agreed with the size obtained by primerextension.

The cDNA sequences of this invention can be further utilized to screenhuman genomic cosmid or phage libraries to isolate human genomicsequences encoding human lipocortin-like polypeptides.*

These human cDNA and genomic sequences can be used to transformeukaryotic and prokaryotic host cells by techniques well known in theart to produce human lipocortin-like polypeptides in clinically andcommercially useful amounts.

It should also be understood that the cDNA sequences of the inventionmay be contained in larger mRNA species which result from alternatesplicing. Such mRNAs may encode a signal sequence for lipocortin inaddition to the mature protein.

D. EXPRESSION OF A LIPOCORTIN PROTEIN IN E.COLI

Plasmid pKK233.LIP.1 (which contains a partial sequence of thelipocortin coding region) was constructed by a three part ligation usingNcoI-Pst -cut pKK233-2 [E. Amann et al., "Vectors Bearing A HybridTrp-Lac Promoter Useful For Regulated Expression Of Cloned Genes InEscherichia coli", Gene, 25 pp. 167-78 (1983)] and the BglII-PstI andNciI-BglII fragments from pL9/20 (see FIG. 5). Plasmid pL9/20 containsthe DNA sequence of nucleotides 67-1376 of the cDNA insert of λLC shownin FIG. 4 inserted into the EcoRI site of pUC13.

Transformants resulting from this ligation and subsequent transformationinto E.coli strain HB101 I^(Q) were picked into microtiter wellscontaining L broth plus ampicillin and grown overnight. The overnightcultures were then replicated onto nitrocellulose filters on L brothagar plus ampicillin plates in quadruplicate and incubated forapproximately 4 h at 37° C. The nitrocellulose filters were thentransferred to L broth plates containing IPTG (10 μg/ml) and incubatedfor 0, 30, 60, or 120 min, followed by lysozyme-detergent treatment tolyse the colonies and finally by Western blot analysis with a crossreactive antiserum that was prepared against the rat lipocortin.Transformants were also analyzed by plasmid restriction mapping. All theWestern positive colonies contained plasmids carrying the predictedrestriction fragments. Preparations of E.coli from the positive colonieswere also analyzed by SDS polyacrylamide gel electrophoresis. WithWestern blot analysis of these preparations using the antibody againstrat lipocortin, we detected a 31,000 molecular weight truncated protein.

We have also constructed various expression vectors in E.coli for theproduction of the full length human lipocortin. All are perfectconstructs starting with the first methionine in the sequence depictedin FIG. 4. We confirmed expression by the procedure described above forthe truncated protein, using an antiserum prepared against ratlipocortin.

For example, FIG. 6 depicts plasmid pLiptrc155A, a trc expression vectorderived from plasmid pKK233-2 [E. Amann et al., supra]. pLiptrc155A hasa hybrid promoter which contains the -10 region from lac and the -35region from trp. It also contains the 5S RNA T₁ T₂ terminators and theβ-lactamase gene which confers ampicillin resistance.

pLiptrc155A was constructed as follows: Plasmid pKK233-2 was restrictedwith NcoI and HindIII, yielding a linear fragment. Plasmid pL9/20 waspartially digested with HindIII and then completely digested with EcoRIand the 1090 fragment was isolated by agarose gel electrophoresis. Thesetwo fragments were then completely digested ligated in the presence of aNcoI-EcoRI linker containing the initiation ATG and the sequence codingfor five amino acids 5' to the EcoRI site in the human lipocortin cDNA.

The resulting pLiptrc155A expression vector was then used to transformE.coli strains JA221 and W3110I^(Q) and expression was induced by growthof the transformed strains for 4 h in LB medium containing 1 mM IPTG and35 μg/ml ampicillin. SDS polyacrylamide gel analysis of crude lysates ofthe transformed host cells showed a single new protein band at anapparent molecular weight of 37 Kd. Control extracts from the strainsnot transformed with pLiptrc155A, or strains transformed with theplasmid but suppressed for production of the protein, did not show this37 Kd protein. We found, for example, that when the JA221 host wastransformed with pLiptrc155A, the 37 Kd protein accounted for as much as2% of the total protein.

To further verify that we were expressing the human lipocortin, the samelysates were also subjected to Western blot analysis using antibodyraised against the rat lipocortin. Only the 37 Kd protein wasimmunoreactive with the antibody against the rat protein. We have alsoshown by Western blot analysis that the natural human lipocortin fromU937 cells (detected by its immunoreactivity with the anti-rat proteinantibody) is virtually identical in size with the 37 Kd protein weexpressed, banding in the same place on the gel.

Finally, a small amount of the expressed protein was electroluted out ofan SDS polyacrylamide gel and subjected to N-terminal sequence analysis.The amino acid sequence obtained was consistent with the predicted aminoacid sequence of the λLC cDNA sequence of FIG. 4.

The human lipocortin which we expressed inhibited exogenousphospholipase A2 in the in vitro assay described in Example A above.This inhibition was detected first using crude lysates and later with amore purified preparation of expressed protein. When the solublefraction of crude lysates prepared with a french pressure cell wasassayed for phospholipase inhibitory activity, we obtained the resultsshown in Table I below. Inhibitory activity was detected in E.colilysates containing plasmid pLiptrc155A, while no activity was detectedin lysates from E.coli that did not contain the plasmid. As determinedby gel analysis, the only difference between these two extracts was thepresence of the 37 Kd protein in the inhibitory fraction. We found thatthe 37 Kd protein accounted for less than 1% of the total protein in thelysate. We also obtained similar results when sonicated lysates wereassayed.

Although inhibitory activity could be detected directly in the solublelysate, most of the 37 Kd protein in E.coli was insoluble and henceremoved by low speed centrifugation after the cells were lysed with thefrench press. The insoluble protein was extracted from particulatematter with guanidine hydrochloride, dialyzed against 1M urea, and thenassayed for phospholipase inhibitory activity. The results of this assayare shown in Table II below. The dialysate contained approximately 200 Uof inhibitory activity per ml (1 U inhibits 15 ng A2). To insure thatthis activity was the result of a protein, and not some other componentin the extract such as lipid, 25 μl of the lysate used in Table II wereincubated with trypsin. As shown in Table III below, the inhibitoryactivity was very trypsin-sensitive.

Table I Phospholipase Inhibit Activity in Crude E. coli Lysates.

Cultures of the W31110I^(Q) strain of E. coli, which either did or didnot contain the plasmid pLiptrc155A, were induced with IPTG and lysedwith a french pressure cell. Particulate matter was removed bycentrifugation at 10,000×g for 20 min. The soluble fraction was assayedfor phospholipase inhibitory activity. The numbers shown are theaverages from several assays in which 50 μl of extracts were assayedwith 100 ng of porcine pancreatic phospholipase A₂.

    ______________________________________                                                              Percent                                                 Sample                Inhibition                                              ______________________________________                                        A.sub.2 alone         0                                                       A.sub.2 + E. coli, no plasmid                                                                       0                                                       A.sub.2 + E. coli containing trc plasmid                                                            24                                                      ______________________________________                                    

Table II Dose-Response Curve Of Partially Purified Inhibitor

The insoluble preparation, which was recovered from JA221 cellstransformed with pLiptrc155A (using the lysis treatment described above)was exposed to 6M guanidine hydrochloride in 25 mM sodium acetate (pH6.0). Particulate matter was removed by centrifugation (100,000,000×gfor 1 h). Extracted protein, which was highly enriched for the human 37Kd protein, was dialyzed against 1M urea in 25 mM sodium acetate (pH6.0) and then assayed for phospholipase A₂ inhibitory activity.

    ______________________________________                                               μl extract                                                                         Percent                                                               assayed Inhibition                                                     ______________________________________                                                0       0                                                                     3      12                                                                    10      26                                                                    30      58                                                             ______________________________________                                    

Table III Trypsin Sensitivity Of Inhibitor.

The partially purified preparation described in Table II was exposed totrypsin for 15 min at room temperature and then assayed with 100 ng ofporcine pancreatic phospholipase A₂. Under the conditions used, thetrypsin treatment did not alter the phospholipase A₂ activity. For eachsample, 25 μ of inhibitor were assayed.

    ______________________________________                                                         Trypsin  Percent                                             Sample           μg/ml Inhibition                                          ______________________________________                                        A.sub.2 alone    0        0                                                   A.sub.2 + inhibitor                                                                            0        54                                                  A.sub.2 + inhibitor                                                                            1        3                                                   A.sub.2 + inhibitor                                                                            3        4                                                   ______________________________________                                    

In addition to pLiptrc155A, we constructed other high level expressionvectors of this invention. For example, plasmid pLipPLT4A wasconstructed as follows' plasmid pPLT4HTNF, a gift from Walter Fiers,(this plasmid is identical to the plasmid deposited in the culturecollection of the Deutsche Sammlung Von Mikroorganismen, in Gottingen,West Germany, on December 27, 1984 under DSM No. 3175 and which wasdeposited within E.coli strain C600 and designated as pBR322-pL-T4-hTNF)was digested with restriction enzymes ClaI and HindIII and a linearfragment was obtained. Plasmid pL9/20 was partially digested withHindIII and then completely with EcoRI, and the 1350 bp fragment wasisolated from an agarose gel. These two fragments were ligated in thepresence of a ClaI-EcoRI linker containing an initiation ATG and thesequence coding for five amino acids 5' to the EcoRI site in thelipocortin cDNA. In the resulting expression vector, the P_(L) promoterdirects the transcription of a hybrid mRNA including sequences of P_(L),T₄ and the lipocortin mRNA. Translation of this mRNA initiates at thefirst ATG of the human lipocortin coding sequence, resulting in a 37 Kdprotein. A second tetracycline resistant plasmid pLipPLT4T wasconstructed by inserting the tetracycline resistance gene of pBR322 intothe ScaI site of pLipPLT4A.

We transformed E.coli strains MCI061 and C600pCi 857 with pLipPLT4A anddetermined expression by SDS polyacrylamide electrophoresis and Westernblot analysis as described above. The E.coli extracts showed a 37 Kdprotein reactive with antibody to rat lipocortin.

E. EXPRESSION OF HUMAN LIPOCORTIN PROTEIN IN YEAST

We have also constructed expression vectors for the production of humanlipocortin in yeast. FIGS. 7-9 show the construction of pBg120 which,when used to transform yeast cells, expressed human lipocortin asdetected by Western blot analysis with the anti-rat lipocortin antibody

We constructed pBg120 as follows:

Plasmid pLXV-1 contains the origin of DNA replication from E.coliplasmid pBR322 and from yeast plasmid 2μ DNA. It can therefore replicatein both E.coli and yeast (J. Ernst, personal communication) We removedthe BamHI site from plasmid pLXV-1 by digestion with BamHI, treatmentwith S1nuclease at 37° C. for 30 minutes and recircularization with T4DNA ligase (FIG. 7). E.coli JA221 was transformed with the ligationmixture and plasmid pLXV-1-BamHI⁻ was isolated.

In order to reconstitute the ATG initiation codon of the acting gene onpLXV-1, we digested pLXV-1-BamHI⁻ with EcoRI and with HindIII. Weisolated the large fragment containing the actin promoter We ligatedtogether this fragment with a DNA fragment containing the human 1antitrypsin gene containing a BamHI end and a HindIII linker* and twosynthetic linkers (linkers 9 & 10).** Linkers 9 and 10 both containedEcoRI and BamHI sticky ends and linker 9 contained an NcoI restrictionsite and the ATG initiation codon. This ligation resulted in the loss ofthe EcoRI and BamHI restriction sites. We transformed E.coli JA221 withthis ligation mixture and selected for Ampicillon resistance andKanamycin resistance to insure the recreation of the HindIII sitelocated within the Kam® gene. We designated the resultant plasmid pAPN.

FIG. 8 shows the insertion of the DNA sequences coding for humanlipocortin into a yeast expression vector. The human lipocortin sequencewas cloned in two parts. First, the N-terminal region was insertedbehind and operatively linked to the yeast actin expression controlsequence. Plasmid pTrp321 (R. Devos et al., "Molecular cloning of humaninterleukin 2 cDNA and its expression in E.coli," Nucleic AcidsResearch, 11, pp. 4307-23 (1983)) was digested with ClaI and HindIII andthe large fragment was ligated to the pL9/20 1090-linker fragmentprepared supra. p. 34. We isolated plasmid pTrplip by screening for theexpression of human lipocortin as described above.

We next digested pAPN with NcoI and isolated the small fragmentcontaining the yeast actin expression control sequence. We ligated thisfragment to pTrp-lip which had been linearized with NcoI. We designatedthis plasmid pTrp-lip™apn-nco. We digested ptrp-lip-apn-nco with HindIIIand isolated the fragment containing the yeast actin expression controlsequence and the DNA sequences corresponding to the N-terminal region ofthe human lipocortin. We ligated this fragment to the large HindIIIfragment of pAPN to produce plasmid pBg114.

We obtained the DNA sequences corresponding to the remaining C-terminalfragment of the human lipocortin from the 800bp BglII-BamHI fragment ofpL9/20, supra (FIG. 9). We digested pL9/20 with BglII and BamHI andelectroeluted the 800bp fragment. This fragment was ligated to pBg114which had been digested with BglII. We isolated plasmid pBg120,containing DNA sequences coding for human lipocortin operatively linkedto the actin expression control sequence.

We detected in extracts of yeast transformed with pBg120 a 37 Kd proteinnot observed in untransformed yeast cells or in yeast cells transformedwith plasmids which did not contain the human lipocortin gene. Thisprotein corresponded exactly on SDS-polyacrylamide gel electrophoresisto the human 37 Kd protein produced in E.coli (FIG. 10).

In yeast the human 37 Kd protein is produced as a soluble protein. Itaccounts for about 4% of the total cellular protein. When yeast culturescontaining the recombinant protein were lysed with a french pressurecell at 20,000 p.s.i. and particulate matter removed by centrifugation(10,000×g, 10 min) approximately 80% of the inhibitory activity wasrecovered in the soluble fraction. The distribution of inhibitoryactivity correlated exactly with the distribution of the human proteinbased on SDS-polyacrylamide gel analysis. The recombinant proteinproduced in yeast has a blocked amino-terminus.

F. EXPRESSION OF HUMAN LIPOCORTIN PROTEIN IN MAMMALIAN CELLS

We have also constructed expression vectors for the production of humanlipocortin in mammalian hosts. FIGS. 11-13 show the construction ofpSVL9109 which, when transfected into cos and CHO host cells by theCaPO₄ procedure (F. Graham et al., J. Virology, 52, pp. 455-56 (1973)),expressed human lipocortin as detected by Western blot analysis with theanti-rat lipocortin antibody. We detected a 37 Kd immunoreactive proteinnot observed in nontransfected cells, indicating that the vector wasproducing the human inhibitor protein. We constructed pSVL9109 asfollows:

As shown in FIG. 11, plasmid pSV2gpt (R. Mulligan et al., Froc. Natl.Acad. Sci. USA, 78, pp. 2072-76 (1981)) was cut with PvuII and HindIIIand the 340 bp fragment containing the SV40 early promoter was isolatedand inserted into pAT153 which had been previously cut with EcoRI, S1treated, and then cut with HindIII. The resulting plasmid pAT.SV2contained the promoter. This plasmid was then cut with HindIII andBamHI. Into this site we cloned a sequence which contained the small tsplice site. This sequence was :solated from another plasmid, pTPA119,by cutting with HindIII and BamHI. The 3 kb insert contained the DNAsequence coding for human tissue plasminogen activator along with thesmall t splice site. This sequence is equivalent to that found in thepTPA25 HindIII-BamHI segment deposited with the American Type CultureCollection in Rockville, Md., on Aug. 21, 1984 under ATCC No. 39808. TheHindIII-BamHI 3 kb piece was ligated to pAT.SV2 to yield plasmidpAT.SV2.TPA. This vector contains the SV40 early promoter followed by aHindIII site and the SV40 small t splice signal preceded by a BglIIsite.

We next inserted the coding sequence for human lipocortin intopAT.SV2.TPA. An oligonucleotide of 29 bp with EcoRI and HindIII ends wassynthesized (see FIG. 12). This oligonucleotide includes the codingsequence for the first six amino acids of human lipocortin. Thissequence was cloned into pUC9 (J. Vieira et al., Gene, 19, pp. 259-68(1982)) which had been digested with EcoRI and HindIII to yield pLP0900.This plasmid was cut with EcoRI and treated with calf alkalinephosphatase. We then cloned the 1.3 kb EcoRI fragment from pL9/20 whichcorresponds to the coding region for human lipocortin into pLP0900. Theresulting plasmid pLP0905 contains the entire coding region for humanlipocortin with a HindIII site upstream (see FIG. 12). This makes thegene suitable for cloning behind the SV40 promoter of pAT.SV2.TPA.

FIG. 13 shows the insertion of the gene for human lipocortin intopAT.SV2.TPA to form a mammalian expression vector of this invention. Thehuman lipocortin sequence was cloned into the expression vector in twoparts. First, the N-terminal region was inserted behind the SV40promoter and then the C™terminal region was added. The plasmid pLP0905was cut with HindIII and BglII and the 560 bp fragment containing theN-terminal region of human lipocortin was isolated. pAT.SV2.TPA was cutwith HindIII and BglII and the 5 kb fragment containing the vector wasisolated, free of TPA sequences. Into this HindIII-BglII vector, weinserted the 560 bp HindIII-BglII fragment containing the N-terminalregion of human lipocortin to yield the plasmid pLP0908.

The C-terminal region of human lipocortin is found within the 800 bpBglII-BamHI fragment of pL9/20. Thus, pL9/20 was cut with BglII andBamHI, followed by electroelution of the 800 bp fragment. This fragmentwas ligated into the plasmid pLP0908 which had been cut with BglII andtreated with calf alkaline phosphatase. Plasmid pSVL9109 was isolated.This plasmid has the entire human lipocortin coding sequence downstreamof the SV40 early promoter followed by the SV40 small t splice signal.Plasmid pSVL9109 was used to transfect cos and CHO hosts as describedabove.

Thus, utilizing the DNA sequences of the invention, we have constructedhigh level expression vectors for the expression of human lipocortin ina biologically active form.

Recombinant DNA sequences prepared by the processes described herein areexemplified by a culture deposited in the culture collection of In VitroInternational, Inc., Ann Arbor, Mich. The culture was deposited on Jan.9, 1985 and is identified as follows:

λLC: IVI No. 10042

Microorganisms prepared by the processes described herein areexemplified by a culture deposited in the above-mentioned depository onMar. 12, 1985 and on Aug. 14, 1985, respectively, and identified asfollows:

E.coli W3110I^(Q) (pLiptrc155A): IVI No. 10046

S.cerevisiae 331-17A (pBg120): IVI No. 10088.

G. PRODUCTION OF BIOLOGICALLY ACTIVE HUMAN LIPOCORTIN-LIKE POLYPEPTIDEFRAGMENTS

The production of polypeptide fragments smaller than the 37,000molecular weight human lipocortin which display phospholipase inhibitoryactivity is highly desirable. A smaller polypeptide is more easilydelivered to target cells by, e.g., transdermal infusion rather thanintravenous injection. A smaller polypeptide might also have a morestable conformation than the 37K protein. For example, the N-terminalend of the 37K protein contains a cluster of hydrophobic amino acids,which may cause the formation of intermolecular aggregates, while theC-terminal end contains four cysteines capable of forming improperdisulfide linkages (see FIG. 4). Thus removal of the N-terminal andC-terminal ends of the large protein may avoid conformationalheterogeneity when the protein is produced in high concentrations.Isolation of biologically active fragments will also permit bettercharacterization of the active site of the lipocortin molecule and leadto the design of fragments with optimal therapeutic value.

Although a variety of methods can be used for generation of polypeptidefragments from their parent molecule in accordance with this aspect ofour invention, we chose to subject the human lipocortin-like polypeptideto limited protease digestion in the illustrative embodiment describedbelow. We first optimized digestion conditions for each protease so thatthe fragments generated were few in number, usually less than ten, andtherefore easily isolated. The optimum conditions were in generaldetermined by the following factors: (1) the type of protease used; (2)the physical state of the target protein, i.e., denaturing ornon-denaturing conditions; (3) the time period for digestion; (4) thetemperature; (5) the amount of protease used; and (6) the pH.

We used two different types of proteases to digest our 37Klipocortin-like polypeptide. The first type hydrolyzes peptide bondsnon-specifically. This type is represented by elastase and proteinase Kproteases. The second type, represented by the proteases plasmin andthrombin, hydrolyzes a specific peptide bond. This second type probablyrecognizes target proteins both by amino acid sequence and byconformation surrounding the cleavage sites. We also used trypsin, athird type of protease, to localize the C-terminal end of the activepeptide fragments (see pp. 48-49, infra). Trypsin hydrolyzes peptidebonds at specific amino acids. Other proteases of this type includechymotrypsin and V8 protease. Because we wished to obtain fragments withbiological activity, we performed digestions under non-denaturingconditions.

1. Production, Isolation And Characterization Of Plasmin-DigestedFragments From Recombinant Human Lipocortin

In order to optimize conditions for plasmin digestion, we incubated analiquot of our E.coli-produced human lipocortin-like polypeptide (100μg) in 100 μl of 0.2M Tris HCl (pH 8.0), 5 mM EDTA and 0.1 mg bovineserum albumin/ml solution with 10 μl (1 μg/μl) plasmin (Calbiochem) atroom temperature. At different time intervals, 15 μl were withdrawn andthe reaction quenched by adding 5 μl of buffer containing 8% SDS andthen boiling the aliquots for 5 min. The digestion mixture at each timepoint was analyzed by SDS gel electrophoresis. The results, depicted inFIG. 14, show that after an hour of incubation almost all the proteinwas digested to a fragment with a molecular weight of 33,000. This 33Kfragment was resistant to further digestion either with longerincubation times or in the presence of increased amounts of plasmin.

Using the digestion conditions described supra. we incubated 500 μg ofhuman lipocortin-like polypeptide with 75 μg of plasmin for 1 hour. Thereaction was stopped with 250 KIU of the protease inhibitor aprotinin(Sigma). We loaded the digested samples onto a Biogel P-60 column(1.0×50 cm), equilibrated with 0.2M Tris-HCl, pH 7.5, 5 mM EDTA, 0.1%bovine serum albumin at 4° C. We collected 1.1 ml fractions. The elutionprofile of fragments monitored at 280 nm disclosed two protein peaks(FIG. 15). We analyzed aliquots of fractions from each peak by highpressure liquid chromatography and by SDS gel electrophoresis. Theresults showed that peak I contained a fragment with 33,000 molecularweight (designated as Lipo-L), and peak II contained a fragment with4,000 molecular weight (designated as Lipo-S).

Fractions of the Biogel P-60 column chromatography were assayed forphospholipase A₂ inhibitory activity, as described supra. The majorityof activity was associated with peak I (FIG. 15). Although a smallamount of inhibitory activity was detected in peak II, that activity wasnot dependent on fragment concentration and was probably caused bynon-specific binding of the fragment to the membrane.

We also isolated the two cleavage products by reverse phase highpressure liquid chromatography with a gradient of acetonitrile from0-75% in 0.1% trifluoroacetic acid using C₄ column chromatography(Vydac). Lipo-S was eluted with 38% acetonitrile and Lipo-L with 48%acetonitrile.

We determined the N-terminal amino acid sequence of Lipo-L (33K; peak I)by sequential Edman degradation using a gas phase sequencer (AppliedBiosystems 470A). PTH-amino acids were analyzed by high pressure liquidchromatography on a 5 μm cyanocolumn (Hypersil) using a gradient ofacetonitrile methanol (4:1) from 15-55% in 0.02M sodium acetate buffer(pH 5.7). We determined the N-terminal amino acid sequence of Lipo-L tobe H₂ N-Gly-Gly-Pro-Gly-Ser-Ala-Val. The N-terminal glycine of Lipo-Lcorresponds to Gly-30 of the 37K protein.

To determine the C-terminal amino acid sequence of our fragments, wecompared the tryptic peptide map of the 37K rat lipocortin (FIG. 16)with a tryptic peptide map which we generated for each of the fragments.We diluted 20 μl of the 37K lipocortin-like polypeptide (40 μg) in 0.2MTris-HCl, pH 8.0, 5 mM EDTA, 0.1 mg bovine serum albumin/ml with 0.2 mlof 0.1M NH₄ HCO₃ plus 1 mM CaCl₂. The mixture was then incubated withtrypsin for 24 hr. at 37° C. . During this incubation, we added trypsinat three time points: 0.5 μg at time zero, 0.25 μg after 4 hr. and 0.25μg after 19 hr. The digestion was stopped by adding 10 μl of 90% formicacid to the reaction mixture. We digested the plasmin fragments usingidentical conditions, except that we first dried down the fragments,which had been purified from high pressure liquid chromatography, with aSpeedvac concentrator (Savant), and dissolved the residues in 0.1M NH₄HCO₃ and 1 mM CaCl₂.

We resolved the tryptic fragments by reverse phase high pressure liquidchromatography with a gradient of acetonitrile from 0-75% in 0.1%trifluoroacetic acid on a C₁₈ column (Spectraphysics). We thendetermined the amino acid sequence of each of the peak fractions. First,we established the amino acid composition of each peak by hydrolyzingthe peptides in 6N HCl for 24 hr. and determining the composition on aBeckman 6300 amino acid analyzer. We compared the observed compositionswith predicted compositions based on sequence data of all possiblefragments from the 37K polypeptide and assigned identifications to eachpeak. We also determined the amino acid sequence of some of the peaksdirectly by sequence analysis.

By comparing the map of fragment Lipo-L (FIG. 17B) with that of 37Kpolypeptide (FIG. 16), we concluded that the biologically active Lipo-Lfragment has the same C-terminal amino acid sequence as that of the 37Kpolypeptide. Therefore, this data, when combined with our N-terminalsequence data, demonstrate that Lipo L contains amino acid residues #30to #346 of the 37K polypeptide. By comparing the peptide maps offragment Lipo S (FIG. 17A) with that of the 37K polypeptide, weconcluded that Lipo S contains amino acid residues #1 to 29 of the 37Kprotein.

2. Production, Isolation And Characterization Of Elastase DigestedFragments From Recombinant Human Lipocortin

We treated 50 μl of the 37K human lipocortin-like polypeptide (75 μg),0.2M Tris-HCl, pH 8.0, 5 mM EDTA, 0.1 mg bovine serum albumin/ml, with2.5 μg (1 μg/l in 10 mM ammonium acetate, pH 6.0) of elastase (Sigma) atroom temperature. We analyzed the digestion products at different timeintervals as described supra to optimize conditions for elastasedigestion. The results showed that 20 min. of incubation generatedfragments with molecular weights ranging from 10K to 33K.

We incubated 150 μg of the 37K polypeptide with 5 μg of elastase underoptimum conditions as described above and subjected the digestedpolypeptides to SDS-polyacrylamide gel electrophoresis (D. A. Hager andR. R. Burgess, Anal. Biochem., 109, 76-86 (1980)). The polypeptide bandswere visualized by soaking the gel in 0.25M KCl, 2mM DTT at 4° C. for5-10 min. We then cut out the polypeptide bands and removed the KCl fromthe gel slices by soaking each slice in deionized H₂ O containing 2mMDTT for 10 min. at room temperature. The fragments were then eluted fromthe gel at room temperature for 2 hr. by a solution of 0.1% SDS; 0.2MTris HCl, pH 7.5; 5 mM EDTA; 5 mM DTT; 0.01% bovine serum albumin. Thefragments-containing solution was then mixed with a solution of ice-coldacetone at -70° C. and incubated for 30' at -70° C. . We collected theprecipitate by centrifugation at 10,000 rpm (SS34 rotor) for 10 min. at4° C. , and rinsed once with ice cold 80% acetone and 20% buffercontaining 0.2M Tris HCl, pH 7.5 and 5 mM DTT. The precipitate wascollected by centrifugation and dried under vacuum for 5-10 min. We thendissolved the dried powder in 6M guanadine hydrochloride in 0.2M TrisHCl (pH 7.9), 2 mM DTT, 5 mM EDTA, 0.1% bovine serum albumin for 20 min.To the solution we then added a "renaturation" buffer (20% glycerol and80% 0.2M Tris-HCl (pH 7.9), 2 mM DTT, 5 mM EDTA, 0.1% bovine serumalbumin). The solution was then allowed to stand at 4° C. overnight. Wenext assayed for phospholipase A₂ inhibitory activity as described. Theactivities of the SDS-gel fragments are shown in FIG. 18. The fragmentse-1, e-2, e-3, e-4 and e-5, with molecular weights of 33K, 30K, 27K, 20K and 18K, respectively, showed phospholipase A₂ inhibitory activity.

We then purified the major active fragments (e-1, e-4, and e-5 of FIG.18) by preparative SDS-gel electrophoresis. We visualized thepolypeptide bands by soaking the gel in 0.25M KCl and excised thebiologically active polypeptide bands. We electroeluted the fragmentsusing standard techniques (M. W. Hunkapiller, E. Lujan, F. Ostrader andL. E. Hood, Methods Enzymol. 91, pp. 227-236 (1983)).

We determined the N-terminal and C-terminal amino acid sequences of e-1,e-4 and e-5. The purified fragments all showed the identical N-terminalamino acid sequence: H₂ N-Gly-Gly-Pro-Gly-Ser-Ala-Val-Ser-Pro-Tyr. ThisN-terminal glycine corresponds to Gly-30 of the 37K protein.

FIG. 19 shows the tryptic peptide maps of these purified fragments. Wecompared these maps with that of the 37K protein (FIG. 16) and foundthat the 18K e-5 fragment lacked the tryptic fragmentAla-Leu-Tyr-Glu-Ala-Gly-Glu-Arg (residues #205-212 of the 37K protein)and all fragments beyond this peptide. This suggests that cleavage isbefore this peptide. The peptide map contains the fragmentAsn-Ala-Leu-Leu-Ser-Leu-Ala-Lys (residues #178-185 of the 37K protein).Thus, the C-terminus of this fragment is between Lys-185 and Arg-212 ofthe 37K protein. Similarly, the peptide map of the 20K e-4 fragmentcontains all the fragments found in the 18K fragment plus an additionalpeptide Ala-Leu-Tyr-Glu-Ala-Gly-Glu-Arg-Arg-Lys (residues #205-214 ofthe 37K protein). The C-terminal end of this fragment is thereforebetween Lys-214 and Arg-228 of the 37K protein. The C-terminal end ofthe 33K e-1 fragment is located between Lys-337 and Asn-346 of the 37Kprotein.

Thus, we have isolated at least three biologically active peptidefragments of the 37K lipocortin-like polypeptide. All three fragmentshave identical N-terminal ends, beginning with Gly 30 of the 37Kpolypeptide, each ending at a different point along the 37K molecule.All three show the phospholipase A2 inhibitory activity of the full-sizelipocortin-like polypeptide.

Although we used the above methods for production of polypeptidefragments from human lipocortin-like polypeptide produced in E.coli, itshould be understood that such molecules can also be produced by avariety of other methods. For example, such molecules can be produced byproteases other than the ones we used or by chemicals which cleave orsynthesize peptide bonds. Furthermore, such molecules can also beproduced by expression of truncated DNA sequences coding for thesefragments. However, this method may require different purificationprocedures for each fragment and occasionally results in the productionof DNA sequences which are unstable in the expression vector.

H. THE PRODUCTION OF BIOLOGICALLY ACTIVE HUMAN LIPOCORTIN-LIKEPOLYPEPTIDE FRAGMENTS BY RECOMBINANT DNA TECHNIQUES

An alternative method of generating lipocortin-like polypeptidefragments by recombinant DNA techniques is to introduce into the DNAsequence encoding lipocortin codons which, upon expression of the DNAsequence in the form of the polypeptide, result in sites at which thepolypeptide can be cleaved (e.g., chemically or proteolytically). Thesecodons can be introduced into the DNA by various techniques known in theart such as mutagenesis or insertion. Thus, an altered lipocortin-likepolypeptide is produced by the host cell, purified in the same manner asthe unaltered full length polypeptide and cleaved to yield a desiredfragment or fragments. Using this strategy, we have altered thelipocortin DNA sequence of the present invention to produce alteredpolypeptides which when cleaved in vitro. yield lipocortin-likepolypeptide fragments displaying phospholipase A₂ inhibitory activity.

According to one embodiment of this technique, we constructed DNAsequences that code for altered lipocortin-like polypeptides bymodifying the DNA sequence of lipocortin to introduce methionine codonsinto the sequence. Upon expression of this sequence in a host cell,altered lipocortin-like polypeptides were produced having either aspecific amino acid replaced by a methionine or an inserted methionineat a particular site along the polypeptide sequence. Treatment of thesealtered polypeptides with cyanogen bromide, which cleaves polypeptidesspecifically at methionine residues, yielded lipocortin-like polypeptidefragments having phospholipase A₂ inhibitory activity.

We have found that the natural human lipocortin protein has only twomethionine residues (at amino acids 56 and 127) which are containedwithin the biologically active elastase fragments described in ExampleG(2) above. Since cleavage of a lipocortin-like polypeptide withcyanogen bromide inactivated the polypeptide and any fragments producedthereby, we inferred that one or both of these methionines werenecessary for the biological activity of the protein. Thus, apreliminary step in the production of active lipocortin-like polypeptidefragments by cyanogen bromide treatment required the replacement ofthese two methionines with another amino acid without destroying thebiological activity of the lipocortin protein. We therefore replacedmethionines 56 and 127 with leucine residues and obtained a biologicallyactive polypeptide fragment upon treatment with cyanogen bromide.

We replaced methionines 56 and 127 by mutagenesis of the DNA sequenceencoding lipocortin as follows: We restricted plasmid pLiptrc155A, whichcontains the DNA sequence encoding lipocortin, with the restrictionenzyme PvuI, resulting in a linearized pLiptrc155A molecule. We alsorestricted plasmid pKK233-2 with the restriction enzymes NcoI andHindIII, which cut the plasmid on either side of the DNA sequenceencoding lipocortin (see Example D and FIG. 6 for the construction ofthese plasmids). Mixture of the two restricted plasmids, when denaturedand reannealed, yielded a gapped heteroduplex with the DNA sequenceencoding lipocortin in an approximately 1300 base pair single-strandedregion. Mutagenic oligonucleotides Lipo 60 and 61 were phosphorylated atthe 5' end and a 320-fold excess was added to the heteroduplex mixture.Lipo 60 and 61 contain DNA sequences similar to a portion of thelipocortin DNA sequence but having specific nucleotide changes such thatexpression of the oligonucleotide sequences results in the replacementof met 127 and met 56 of lipocortin with a leucine residue, respectively(see FIG. 20). These oligonucleotides therefore hybridize to thesingle-stranded region of the heteroduplex molecules. The DNA gaps inthe heteroduplex molecules were filled in with the Klenow fragment ofDNA polymerase I and the DNA ligated with T4 polynucleotide kinase [see,Morinaga, et al., Biotechnology, 2, pp. 636-39 (1984 )]. We transformedE.coli strain JA221 with this DNA and selected for transformants byculturing the host cells on LB-ampicillin plates.

We screened the transformants for those colonies containing an alteredlipocortin DNA sequence by hybridization with ³² P-labeledoligonucleotides Lipo 60 and 61 as follows: We transferred the coloniesto a nitrocellulose filter and placed the filter colony side up on afresh LB-ampicillin plate. We incubated the plate for 4 h at 37° C. . Wethen transferred the filter to a fresh LB plate containing ampicillinand chloramphenicol (250 μg/ml) and incubated the plate overnight at 37°C. . We lysed the colonies with 0.5M NaOH-1.5M NaCl and neutralized thelysate twice with 0.5M Tris-HCl (pH 7.7)-1.5M NaCl. We baked the filtersin a vacuum oven for 2 h at 80° C. and prehybridized them in 200 ml of6×SSPE (0.9M NaCl, 90 mM sodium phosphate, 6 mM sodium EDTA, pH 7.0),0.5% SDS, 100 μg/ml tRNA and 5x Denhardt's solution at 55° C. withshaking for several hours. We then hybridized the filters overnight at55° C. in 200 ml of the prehybridization mixture containing 10 pmoles of³² P-labeled oligonucleotide. We washed the filters with 6xSSPE anddetected hybridization by autoradiography [see T. Maniatis, et al.,supra.

We isolated a number of positive clones by this procedure. One of theseclones, designated Lipo 8, was grown up and its plasmid DNA, designatedpLipo8, was extracted and sequenced according to the technique of Maxamand Gilbert, supra. This sequencing analysis showed that the desirednucleotide replacements, i.e., replacement of the DNA codons encodingmet 56 and 127 with codons encoding leucine, had been achieved.

We also extracted the expressed protein from Lipo 8 and treated thispreparation with 3 mg/ml of cyanogen bromide in 70% formic acid toproduce lipocortin-like polypeptide fragments. We tested this crudefragment mixture for phospholipase A₂ inhibitory activity using the invitro assay described earlier. Whereas wild-type lipocortin wasinactivated by cyanogen bromide treatment for 1 h, the fragment mixtureshowed activity after 4 h of treatment. We then isolated a 26 Kdpolypeptide fragment from this mixture by SDS-polyacrylamide gelelectrophoresis (see FIGS. 21 and 22) and renatured the fragment asdescribed in Example G(2) above. We tested this purified fragment forphospholipase A₂ activity using the in vitro assay and found that thislarge fragment of Lipo 8 displayed biological activity.

Once we had demonstrated the ability to replace met 56 and 127 withoutaffecting the biological activity of the resulting lipocortinpolypeptide fragment, we could then replace other amino acid residues ofthe lipocortin protein with methionine or insert methionines intovarious sites within the amino acid sequence of the lipocortin protein.In this way, new cleavage fragments of lipocortin with biologicalactivity were isolated.

For example, using the procedure outlined above, we replaced the leucineat residue 109 of the amino acid sequence of lipocortin with amethionine. We hybridized mutagenic oligonucleotide Lipo 68 (see FIG.20) to a heteroduplex formed from restriction of plasmids pKK233-2 andpLipo8 (which is identical to the pLiptrc155A of FIG. 6 except for thenucleotide changes of Lipo 60 and 61). We thus obtained a transformant,Lipo 11, that contains the desired nucleotide changes to replace Leu 109with a methionine. pLipoll DNA was extracted from this transformant andsequencing indicated the desired nucleotide changes had been achieved.Clone Lipo 11 was then grown up in culture and expressed proteinextracted and treated with cyanogen bromide to produce fragments. Weisolated a 14.6 Kd polypeptide fragment (see FIG. 21) bySDS-polyacylamide gel filtration of the treated protein extract [SeeBiorad Catalog/Price List K, p. 37 (1985)] and assayed this fragment forbiological activity using the in vitro assay. This assay indicated thatthe Lipo 11 fragment possesses phospholipase A₂ inhibitory activity.

Similarly, oligonucleotide Lipo 78 (see FIG. 20) was used to generate atransformant, Lipo 15, which contains the desired nucleotide changes toreplace Leu 198 with a methionine. Cleavage of the extracted expressedprotein with cyanogen bromide followed by SDS-polyacrylamide gelfiltration resulted in the purification of a 20.7K polypeptide fragment(see FIG. 21) which was then assayed for biological activity. This assayindicated that the Lipo 15 fragment possesses phospholipase A₂inhibitory activity.

Alternatively, we inserted a methionine into the lipocortin amino acidsequence at residue 169 by inserting a synthetic oligonucleotide into arestriction site on the lipocortin DNA sequence, thereby creating acodon for methionine at that site. We restricted pLipo8 with BglII andligated into that site oligonucleotides Lipo 75 and 76 (see FIG. 20)using T4 ligase. These complementary oligonucleotides were constructedwith sticky ends complementary to the cohesive ends of the BglII sitefor insertion into the plasmid. We transformed E.coli JA221 cells withthis ligation mixture and selected transformants by growth onLB-ampicillin plate. We screened the transformants for those coloniescontaining the correct orientation of the inserted oligonucleotides byrestriction mapping. One such colony, designated Lipo 9, was grown up inculture and its expressed protein extracted. The crude protein extractwas treated with cyanogen bromide and subjected to SDS-polyacrylomidegel electrophoresis. A 17.7 Kd polypeptide fragment was produced and theextra amino acids introduced into the sequence by insertion of thesynthetic oligonucleotides cleaved away (see FIG. 21).

Thus, we have isolated at least three biologically activelipocortin-like polypeptide fragments by the use of recombinant DNAtechniques--the 26 Kd fragment of Lipo 8, the 14.6 Kd fragment of Lipo11, and the 20.7 Kd fragment of Lipo 15. It is to be understood,however, that other lipocortin-like polypeptide fragments can beproduced by the abovedescribed procedure (e.g., by replacement of otheramino acids along the lipocortin amino acid sequence with methionine).Furthermore, lipocortin-like fragments can be produced by altering theDNA sequence

.61encoding lipocortin to introduce or insert amino acids other thanmethionine to yield an altered polypeptide. This polypeptide can then becleaved by an appropriate enzyme or chemical to yield biologicallyactive fragments (e.g., cysteines can be inserted and the proteincleaved with NTCB, 2-nitro-5thiocyanobenzoic acid).

ISOLATION OF A DNA SEQUENCE ENCODING N-LIPOCORTIN

We have also obtained a nucleotide sequence encoding a portion ofN-lipocortin, a human lipocortin-like polypeptide displayingphospholipase A₂ inhibitory activity and having amino acid homology tothe 37 Kd lipocortin produced according to this invention.

I. PURIFICATION OF LIPOCORTIN AND N-LIPOCORTIN FROM HUMAN PLACENTA

A fresh placenta was packaged on ice immediately after birth andprocessed within 6 h as follows: We skinned the placenta, cut it intocubes and washed the tissue with ten 300 ml changes of ice cold PBS (50mM Na₂ HPO₄, pH 7.2, 150 mM NaCl) and 5% sucrose until the tissue waspink and no additional hemoglobin was released by the washes. The washedplacenta weighed about 350 g.

We washed 30 g of the placenta tissue with 150 ml of extraction buffer(25 mM Tris-HCl, pH 7.7, 5 mM EDTA, 0.1 mg/ml aprotinin, 0.1 mg/mlsoybean trypsin inhibitor, and 40 uM pepstatin A) and then suspended thetissue in 100 ml of the same buffer. We disrupted the preparation with apolytron for 5-7 min on ice and subjected the homogenate tocentrifugation at 4° C. for 15 min at 10,000 rpm in an SS34 rotor. Wedecanted the supernatants, disrupted the pellets with a polytron in anadditional 100 ml of extraction buffer and subjected the suspensionagain to centrifugation at 10,000 rpm for 15 min. The supernatants ofboth extractions were combined and processed.

First, we subjected the extract to DEAE-cellulose chromatography on an80 ml column (DE52, Whatman Ltd., column dimensions: 2.5 cm dia×16 cm)that had been equilibrated with 25 mM Tris-HCl (pH 7.7). We concentratedthe flow-through preparations by ultrafiltration with an Amicon PMlOmembrane to 15 ml. We then subjected the concentrated preparation tocentrifugation for 15 min at 18,000 xg in an SS34 rotor. The apparentionic strength of the concentrate was approximated by measuring theconductivity of the flowthrough from the ultrafiltration. Based on thisvalue, we diluted the concentrate with water to an apparent ionicstrength equal to that of 25 mM Tris-HCl (pH 7.7) (10 ml of water wasadded). We next subjected this preparation to a second DEAE-cellulosestep on a 40 ml column and further fractionated the DEAE-flow-through bygel filtration chromatography on a 200 ml P150 column (columndimensions: 2.5 cm dia×40 cm), which also was performed in 25 mMTris-HCl (pH 7.7). We collected 5 ml fractions of this eluate andassayed an aliquot for phospholipase inhibitory activity using the invitro assay described in Example A above. According to this assay, theeluate contained a single broad peak of inhibitory activity with anapparent molecular weight of 40 K as determined by gel filtration. Wealso characterized an aliquot of the eluate by SDS-polyacrylamide gelelectrophoresis. This gel analysis indicated that the inhibitoryactivity in the eluate corresponded to a prominent protein band at 35Kd. Western blotting analysis demonstrated that the 35 Kd band wasimmunoreactive with a rabbit antiserum against recombinant lipocortinproduced in E.coli.

We next subjected the eluate from the gel filtration column to reversephase high pressure liquid chromatography (HPLC) on a C4 column (Vydac),using a gradient of acetonitrile from 0-75% in 0.1% trifluoroacetic acidto elute the bound fragments. We found that the eluate actuallycontained two 35 Kd components: a natural human lipocortin componentthat eluted from the column with 65.8% acetonitrile and an N-lipocortincomponent that eluted with 65.1% acetonitrile. Only the naturallipocortin component was immunoreactive with the antibody raised againstrecombinant lipocortin.

In order to further purify the two components in a manner in which weretained phospholipase A₂ inhibitory activity, we subjected thepartially purified preparation from the gel filtration column to fastprotein liquid chromatography (FPLC, Pharmacia) on a Mono S highresolution cation exchange chromatography resin (HR5/5, Pharmacia). AtpH 6.0 in 50 mM MES buffer (2-morpholino-ethanesulfonic acid), bothcomponents bound to the Mono S column and were eluted with a gradient ofNaCl. FIG. 23 shows the elution profile of the proteins (absorbancemonitored at 280 nm) (panel A) and the profile of phospholipase A₂inhibitory activity (panel B) from the Mono S column. In panel A, peak Icorresponds to lipocortin and peak II to N-lipocortin. Only the materialin peak I was immunoreactive with the rabbit antiserum againstrecombinant lipocortin. Panel B clearly indicates that both lipocortinand N-lipocortin show phospholipase A₂ inhibitory activity. Based ontheir relative amounts in the purified preparations, we estimate thatthe specific activities of the two proteins as phospholipase inhibitorsare roughly identical.

We have also analyzed these two proteins by tryptic mapping. A largerportion of a placenta was processed and the proteins purified using theprotocol as detailed above. The sizes and volumes of solutions andcolumns were adjusted appropriately. After the two 35 Kd proteins wereseparated by reverse phase HPLC, 100 μg of each of the purified proteinswas subjected to tryptic mapping analysis as follows: We dried theappropriate HPLC fractions under vacuum in a Speed Vac Concentrator(Savant), resuspended the resulting pellets in 400 μl of 0.1 M ammoniumbicarbonate (pH 8.0, 0.1 mM CaCl₂), and digested the mixture withtrypsin. TPCK-trypsin (Worthington, 5 μg total) was added to 100 μg ofprotein and the digestion performed for 16 h at 37° C. The trypsin wasadded in three equal aliquots: the first at time zero, the second after4 h, and the third after 12 h of incubation. The digest was acidifiedwith formic acid to 20% (v/v).

We then resolved the cleavage fragments from the trypsin digestion byreverse phase high pressure liquid chromatography at 40° C. on a C18column as described earlier in Example A. The column eluate wasmonitored at 214 nm. The tryptic maps for lipocortin (panel A) andN-lipocortin (panel B) are shown in FIG. 24. The tryptic map of thenatural human lipocortin isolated from placenta is identical to thetryptic map obtained from the 37 Kd lipocortin derived from theexpression of human macrophage cDNA. DNA sequence analysis revealed twoamino acid differences between the placenta and macrophage-derivedlipocortins. The map for N-lipocortin shows that protein to be unique.

We concentrated the eluate fractions corresponding to eleven of thepeaks from the tryptic map of N-lipocortin on a Speed Vac Concentratorand subjected them to amino-terminal protein sequence analysis using agas phase sequencer (Applied Biosystems 470A). PTH-amino acids from eachcycle of the Edman degradation were analyzed by reverse phase highpressure liquid chromatography using the Applied Biosystems 120A PTHanalyzer. The sequences derived from these analyses are shown in FIG.25. (The "T" numbers to the left of the sequences in FIG. 25 correspondto the peak numbers which are shown on the column profile at the top ofthe figure.)

Most of the peptide sequences of N-lipocortin are unique, although someshow sequence homology to human lipocortin purified in accordance withthis invention, suggesting that the two 35 Kd components--lipocortin andN-lipocortin, respectively --may be derived from the same family ofproteins. As shown in FIG. 25, to date, we have determined the primarystructure of approximately one third of the N-lipocortin molecule. Sincethe sequences of those fragments derived from HPLC peaks containingmultiple fragments can be determined using techniques known in the art,we have actually provided the data necessary to determine the primarystructure of approximately 50% of the molecule.

J. SYNTHESIS OF OLIGONUCLEOTIDE PROBES FOR N-LIPOCORTIN PROTEINSEQUENCES

Having determined the amino acid sequences of various regions ofN-lipocortin from human placenta (see FIG. 25), we chemicallysynthesized four pools of antisense oligonucleotide DNA probes thatcoded for some of those protein sequences. We used the same strategy andprocedure as detailed earlier in Example B for recombinant lipocortin.The amino acid sequences of the four selected regions of N-lipocortinand all the possible nucleotide codon combinations that encode them areshown in FIG. 26. Coding degeneracies are indicated as follows: N=C, T,A or G; R=A or G; Y=C or T; H=A, C or T, P=G or C; X=A, G or T; and Z=Aor T.

As shown in FIG. 26, the four pools of DNA probes correspond to theamino acid sequences of tryptic fragments T20, T25, T24 and T9,respectively. To reduce further the degeneracies in the probes, weprepared each pool in subpools. Oligonucleotides NLipl to NLip3,corresponding to the amino acid sequence of fragment T20, are 24-merswith 128 fold degeneracy. Oligonucleotides NLip4 to NLip7 are 18-merswith 32 fold degeneracy and are homologous to the amino acid sequence offragment T25. Oligonucleotides NLip8 to NLipll are 20-mers with 72 folddegeneracy, corresponding to the amino acid sequence of fragment T24,and NLip12 to NLip15 are 17-mers with 128 fold degeneracy, derived fromthe amino acid sequence of fragment T9.

To test if our synthetic probes actually recognized human sequences, wehybridized the 3 subpools, NLipl, NLip2, and NLip3, which wereend-labeled with ³² P using [γ]-³² P-ATP and T4 polynucleotide kinase,to GeneScreen filters containing poly (A)⁺ mRNA from human placenta,utilizing the Northern blotting technique [H. Lehrach et al., supra].Subpool NLipl hybridized to an 1800 nucleotide mRNA which appears to bethe same size as lipocortin mRNA.

K. CONSTRUCTION AND SCREENING 0F A HUMAN PLACENTA cDNA LIBRARY

We constructed a human cDNA library from poly (A)⁺ mRNA isolated fromhuman placenta using essentially the same procedure as described earlierin Example C for construction of the human macrophage cDNA library. Inthis embodiment, however, we extracted total RNA from the human placentaof a female fetus by the guanidine isothiocyanate method essentially asdescribed by J. M. Chirgwin et al. [Biochemistry, 18, pp. 5294-99(1979)]. This RNA preparation was enriched for poly (A)⁺ RNA by twopassages over an oligo(dT)-cellulose column (PL Biochem) and used tosynthesize double stranded cDNA sequences which were then inserted intoλgt10 and amplified in E.coli C600 hfl cells.

We screened the human placenta cDNA library with the labeledoligonucleotide probe, NLipl, using nitrocellulose filters as describedbelow. An overnight culture of C600 hfl cells in L broth and 0.2%maltose was pelleted and resuspended in an equal amount of SM buffer. Wepre-adsorbed 0.9 ml of cells with 2×10⁶ phage particles at roomtemperature for 15 min. We diluted the suspension to 50 ml in LB plus 10mM MgSO₄ and 0.7% agarose at 55° C. and plated it on LB plates plus 10mM MgSO₄. We screened 30 such plates. We incubated the plates at 37° C.for approximately 5 h and then chilled the plates at 4° C. for 1 h toallow the agarose to harden. We then transferred the λ phage particlesfrom the plaque library plates to S&S nitrocellulose filters by placingthe filters onto the plates containing the recombinant plaques for 5min. We then lifted the filters from the plates, lysed the phages on thefilters by placing the filters onto a pool of 0.5 N NaOH-1.5 M NaCl for5 min and neutralized and submerged the filters in 1 M Tris-HCl-1.5 MNaCl (pH 7.0). The filters were then air-dried and baked at 80° C. for 2h.

We prehybridized and hybridized the treated filters to theoligonucleotide probe, NLipl, in 0.2% polyvinylpyrrolidone (M.W.40,000), 0.2% ficoll (M.W. 400,000), 0.2% bovine serum albumin, 0.05 MTris-HCl (pH 7.5), 1M sodium chloride, 0.1% sodium pyrophosphate, 1%SDS, 10% dextran sulfate (M.W. 500,000) and denatured salmon sperm DNA(≧100 μg/ml) according to manufacturer's specifications (New EnglandNuclear) for Colony/Plaque Screen™ membranes. We detected hybridizingcDNA sequences by autoradiography.

By means of this technique, we picked six positive plaques andrescreened at lower density using the same probe. We isolated the DNA ofthese clones, digested the DNA with EcoRI, and hybridized the sequenceswith the NLipl probe using the Southern blot technique [E. M. Southern,supra]. The cDNA inserts of all six clones hybridized to the NLiplprobe.

We next restricted the DNA of these phages with EcoRI and isolated thecDNA inserts. Digestion of one of the clones, λ-Nlipo21-2, with EcoRIresulted in an approximately 500 base pair fragment which we subclonedinto plasmid pUC9 to produce plasmid pNlipl. We sequenced this plasmidby the method of Maxam and Gilbert [A. M. Maxam and W. Gilbert, supra].As depicted in FIG. 27, the plasmid carries a 394 base pair cDNA insertthat contains 99 base pairs of the N-lipocortin coding sequence(corresponding to 33 amino acids), and 295 base pairs of theN-lipocortin 3' non-coding region, including a poly (A) addition siteand an approximately 50 base pair poly (A) tail. The DNA coding sequenceof N-lipocortin not only contains the sequence corresponding to theoligonucleotide probe derived from tryptic fragment T20, i.e., NLipl,but also the sequence corresponding to tryptic fragment T32 (see FIG.25), thus confirming that we have cloned a portion of the gene encodingthe N-lipocortin protein. This cDNA sequence as well as theoligonucleotide probes described in Example J can then be used as probesto screen for and isolate the DNA sequence encoding full lengthN-lipocortin.

Although clone λ-Nlipo21-2 contains the N-lipocortin DNA sequenceencoding only a small region of the protein, the structural homologybetween lipocortin and N-lipocortin is borne out by this region. Asshown in Table 4 below, 11 of the last 17 amino acids at the carboxylterminus of the two proteins are identical. Of the five differences,most are conservative amino acid substitutions. Based on the similarityin the phospholipase A₂ inhibitory activity of the two proteins and thesimilarity in the protein and DNA sequences of the proteins, we concludethat N-lipocortin and lipocortin represent a family of related proteins.

                  TABLE IV                                                        ______________________________________                                        SEQUENCE HOMOLOGY AT C-TERMINUS OF                                            LIPOCORTIN AND N-LIPOCORTIN                                                   ______________________________________                                        LIPO:GlnLysMetTyrGlyIleSerLeuCysGlnAlaIleLeu                                   ##STR1##                                                                     LIPO:AspGluThrLysGlyAspTyrGluLysIleLeuValAla                                   ##STR2##                                                                     LIPO:LeuCysGlyGlyAsn                                                           ##STR3##                                                                     ______________________________________                                    

A comparison of the nucleotide sequences of the lipocortin andN-lipocortin of this invention shows approximately 60% homology (seeFIG. 28).

Microorganisms and recombinant DNA sequences prepared by the processesdescribed herein are exemplified by a culture deposited in the culturecollection of In Vitro International, Inc., Ann Arbor, Mich. The culturewas deposited on Jan. 8, 1986 and is identified as follows:

E.coli JA221(pNLipl):IVI No. 10093

IMPROVING THE YIELD AND ACTIVITY OF HUMAN LIPOCORTIN-LIKE POLYPEPTIDESPRODUCED IN ACCORDANCE WITH THIS INVENTION

The level of production of a protein is governed by three major factors:the number of copies of its gene within the cell, the efficiency withwhich those gene copies are transcribed and the efficiency with whichthey are translated. Efficiency of transcription and translation (whichtogether comprise expression) is in turn dependent upon nucleotidesequences normally situated ahead of the desired coding sequence. Thesenucleotide sequences or expression control sequences define, inter alia,the location at which RNA polymerase interacts to initiate transcription(the promoter sequence) and at which ribosomes bind and interact withthe mRNA (the product of transcription) to initiate translation. Not allsuch expression control sequences function with equal efficiency. It isthus of advantage to separate the specific lipocortin coding sequencesof this invention from their adjacent nucleotide sequences and to fusethem instead to other known expression control sequences so as to favorhigher levels of expression and production of lipocortin-likepolypeptides. This having been achieved, the newly engineered DNAfragments may be inserted into higher copy number plasmids orbacteriophage derivatives in order to increase the number of gene copieswithin the cell and thereby further to improve the yield of expressedlipocortin-like polypeptides.

Several expression control sequences may be employed as described above.These include the operator, promoter and ribosome binding andinteraction sequences (including sequences such as the Shine-Dalgarnosequences) of the lactose operon of E.coli ("the lac system"), thecorresponding sequences of the tryptophan synthetase system of E.coli("the trp system"), the major operator and promoter regions of phage λ(O_(L) P_(L) as described above and O_(R) P_(R)), a control region offilamentous single-stranded DNA phages, the tac or trc system, thepromoter for 3-phosphoglycerate kinase or other glycolytic enzymes, thepromoters of acid phosphatase, e.g., Pho5, the promoters of the yeastα-mating factors, promoters for mammalian cells such as the SV40 earlyand late promoters, adenovirus late promoter and metallothioninepromoter, and other sequences which control the expression of genes ofprokaryotic or eukaryotic cells and their viruses or combinationsthereof.

Therefore, to improve the production of the lipocortin-like polypeptidesof this invention, the DNA sequences for those polypeptides may beprepared as before and inserted into a recombinant DNA molecule closerto its former expression control sequence or under the control of one ofthe above improved expression control sequences. Such methods are knownin the art.

Other methods useful to improve the efficiency of translation involvethe insertion of chemically or enzymatically prepared oligonucleotidesin front of the initiating codon of the lipocortin-related DNA sequencesof this invention or the replacement of codons at the N-terminal end ofthe DNA sequence with those chemically or enzymatically preparedoligonucleotides. By this procedure, a more optimal primary and higherorder structure of the messenger RNA can be obtained. More specifically,a sequence can be so designed that the initiating AUG codon occurs in areadily accessible position (i.e., not masked by secondary structure)either at the top of a hairpin or in other single-stranded regions. Theposition and sequence of the aforementioned Shine-Dalgarno segment cansimilarly be optimized. The importance of the general structure(folding) of the messenger RNA has been documented (D. Iserentant and W.Fiers, "Secondary Structure Of mRNA And Efficiency Of TranslationInitiation", Gene, 9, pp. 1-12 (1980)).

Further increases in the cellular yield of the lipocortin-likepolypeptides of this invention may be achieved by increasing the numberof genes that can be utilized in the cell. This may be achieved byinsertion of the lipocortin gene (with or without its transcription andtranslation control elements) into a higher copy number plasmid or intoa temperature-controlled copy number plasmid (i.e., a plasmid whichcarries a mutation such that the copy number of the plasmid increasesafter shifting the temperature (B. Uhlin et al., "Plasmids WithTemperature-Dependent Copy Number For Amplification Of Cloned Genes AndTheir Products", Gene, 6,pp. 91-106 (1979)).

Alternatively, an increase in gene dosage can be achieved, for example,by insertion of recombinant DNA molecules, engineered in the mannerdescribed above, into the temperate bacteriophage λ, most simply bydigestion of the plasmid with a restriction enzyme, to give a linearmolecule which is then mixed with a restricted phage λ cloning vehicle(e.g., of the type described by N. E. Murray et al., "Lambdoid PhagesThat Simplify The Recovery Of In Vitro Recombinants", Mol. Gen. Genet.150, pp. 53-61 (1977), and N. E. Murray et al., "Molecular Cloning OfThe DNA Ligase Gene From Bacteriophage T4", J. Mol. Biol., 132, pp.493-505 (1979)), and the recombinant DNA molecule produced by incubationwith DNA ligase. The desired recombinant phage is then selected and usedto lysogenize a host strain of E.coli.

Therefore, it should be understood that the lipocortin-like polypeptidecoding sequences of this invention may be removed from the disclosedvectors and inserted into other expression vectors, as previouslydescribed (supra) and these vectors employed in various hosts, aspreviously described (supra) to improve the production of the humanlipocortin-like polypeptides of this invention.

While we have hereinbefore presented a number of embodiments of thisinvention, it is apparent that our basic construction can be altered toprovide other embodiments which utilize the processes and compositionsof this invention. Therefore, it will be appreciated that the scope ofthis invention is to be defined by the claims appended hereto ratherthan by the specific embodiments which have been presented hereinbeforeby way of example.

We claim:
 1. An essentially pure fragment of human lipocortin, selectedfrom the group consisting of(a) Lipo-L (b) e-1, (c) e-2, (d) e-3, (e)e-4, (f) e-5, (g) the 26 Kd fragment of Lipo 8, (h) the 14.6 Kd fragmentof Lipo 11, and (i) the 20.7 Kd fragment of Lipo
 15. 2. Apharmaceutically acceptable composition useful in the treatment ofarthritic, allergic, dermatologic, ophthalmic, and collagen diseases andother disorders involving inflammatory processes which comprise apharmaceutically effective amount of at least one polypeptide selectedfrom the group consisting of polypeptides according to claim
 1. 3. Amethod for treating arthritic, allergic, dermatologic, ophthalmic, andcollagen diseases and other disorders involving inflammatory processeswhich comprises administering a pharmaceutically effective amount of acomposition according to claim
 2. 4. A method for treating arthritic,allergic, dermatologic, ophthalmic, and collagen diseases and otherdisorders involving inflammatory processes which comprises administeringa pharmaceutically effective amount of an N-lipocortin, the N-lipocortinhaving the following characteristics:(a) its ability to inhibit theenzyme phospholipase A₂, (b) comprising the tryptic fragments:(1)Leu,Tyr,Asp,Ser,Met,Lys; (2) Leu,Ser,Leu,Asn,Gly,Asp,Thr,Ser,Thr,Pro,Pro,Ser,Ala,Tyr,Gly; (3) Ser,Leu,Tyr,Tyr,Tyr,Ile,Gln,Gln,Asp,Thr,Lys; (4) Trp,Ile,Ser,Ile,Met,Thr,Gla,Arg; (5)Ser,Tyr,Ser,Pro,Tyr,Asp,Met,Leu,Glu, Ser,Ile; (6)Leu,Leu,Val,Val,Tyr,Pro,X,Thr,Gln, Ile,Leu;wherein X is any amino acid,and (c) having a carboxy-terminus consisting of the amino acid sequence:LysArgLysTyrGlyLysSerLeuTyrTyrIleGlnGlnAspThrLysGlyAspTyrGlnLysAlaLeuLeuTyrLeuCysGlyGlyAspAsp, the N-lipocortin being selectedfrom the group consisting of:(1) a mature N-lipocortin; and (2) amet-N-lipocortin.
 5. A method for reducing inflammation which comprisesadministering a pharmaceutically effective amount of an N-lipocortin,the N-lipocortin having the following characteristics:(a) its ability toinhibit the enzyme phospholipase A₂, (b) comprising the trypticfragments:(1) Leu,Tyr,Asp,Ser,Met,Lys; (2)Leu,Ser,Leu,Asn,Gly,Asp,Thr,Ser,Thr, Pro,Pro,Ser,Ala,Tyr,Gly;(3)Ser,Leu,Tyr,Tyr,Tyr,Ile,Gln,Gln,Asp, Thr,Lys; (4)Trp,Ile,Ser,Ile,Met,Thr,Glu,Arg; (5)Ser,Tyr,Ser,Pro,Tyr,Asp,Met,Leu,Glu, Ser,Ile; (6)Leu,Leu,Val,Val,Tyr,Pro,X,Thr,Gln, Ile,Leu;wherein x is any amino acid,and (c) having a carboxy-terminus consisting of the amino acid sequence,LysArgLysTyrGlyLysSerLeuTyrTyrTyrIleGlnGlnAspThrLysGlyAspTyrGlnLysAlaLeuLeuTyrLeuCysGlyGly AspAsp; theN-lipocortin being selected from the group consisting of:1. a matureN-lipocortin; and
 2. a met-N-lipcocortin.